Cell scaffold matrices with incorporated therapeutic agents

ABSTRACT

The invention is directed to methods and compositions for preparing matrices for controlled delivery of at least one therapeutic or biological agent to a target site in a subject. This is accomplished using nanoparticles coupled to the therapeutic or biological agent that are incorporated within the matrix or reacted on the surface of the matrix.

RELATED APPLICATION

The present application claims priority to a provisional applicationentitled “Electrospun Cell Matrices” filed on Mar. 11, 2005 and havingSer. No. ______(Atty Dkt No. 105447-3).

BACKGROUND OF THE INVENTION

The technical field of this invention relates to matrices for cellularattachment and growth. The invention also relates to methods of makingand using these matrices for tissue engineering and the construction ofartificial structures.

Synthetic matrices have been used for artificial tissue construction.However, these synthetic matrices often elicit an adverse immuneresponse in a patient. To circumvent this problem, decellularizedmatrices have been used. These decellularized matrices are advantageousfor several reasons. They are naturally derived, and therefore lesslikely to induce an adverse immune response. They also have a similarcomposition, ultrastructure and biomechanics to the native tissue. Whiledecellularized matrices are a promising as scaffolds, they are onlyavailable in a limited supply.

In addition, problems may arise once the artificial tissue construct isdelivered to a target site in the patient, such as an adverse immuneresponse due to the matrix used for the construct, or the requirementfor rapid vascularization so that the artificial construct can continueto grow and develop. To circumvent these problems, therapeutic agentsare typically provided systemically the patient. However, this is oftenleads to serious side effects. Other methods involve localizedinjections of the therapeutic agents immediately after implantation andover the course of several weeks after implantation. Patient complianceis typically poor because multiple injections are needed, with as oftenas three injections per day.

Accordingly, a need exists for creating improved matrices for tissueengineering. In particular, a need exists for creating matrices that canlocally deliver therapeutic agents to a target region.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions for preparingmatrices for cellular attachment and growth. These matrices can also beused for controlled delivery of at least one therapeutic or biologicalagent to a target site in a subject. The controlled delivery isaccomplished using nanoparticles, such as quantum dots, that are coupledwith a therapeutic or biological agent. The therapeutic or biologicalagent can then be releases at the target site by applying radiationenergy at a particular wavelength. This allows the matrix with cells anda therapeutic agent to be implanted at a target site in a subject. Then,the release of the therapeutic agent can be controlled at the targetsite by breaking the bond between the nanoparticle and therapeutic orbiological agent, with radiation energy

Accordingly, in one aspect of the invention, the invention pertains toan electrospun matrix having a three-dimensional ultrastructure ofinterconnected fibers and pores to permit cell attachment and furthercomprising nanoparticles incorporated within the matrix.

The nanoparticles can be quantum dots made selected from the groupconsisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP, GaAs, GaSb,InP, InAs, InSb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge, Si andcombinations thereof. Preferred quantum dots are CdSe quantum dots.

These nanoparticles can be coupled to a therapeutic agent such as growthfactors, proteins, antibodies, nucleic acids molecules, andcarbohydrates. A preferred therapeutic agent is a growth factor selectedfrom the group consisting of transforming growth factor-alpha (TGF-α),transforming growth factor-beta (TGF-β), platelet-derived growth factor(PDGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brainderived neurotrophic factor, cartilage derived factor, bone growthfactor (BGF), basic fibroblast growth factor, insulin-like growth factor(IGF), vascular endothelial growth factor (VEGF), granulocyte colonystimulating factor (G-CSF), hepatocyte growth factor, glial neurotrophicgrowth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor(KGF), and skeletal growth factor. A preferred therapeutic agent isheparin. The therapeutic agent can be encapsulated in a polymer with thenanoparticle.

To release the therapeutic agent from the nanoparticle, radiation energyof a particular wavelength can be applied. The radiation causeslocalized heating of the nanoparticles which induces structural changesin the polymer to release the therapeutic agent. Suitable wavelength ofthe radiation can be between 700-1000 nanometers.

The electrospun matrix can further comprise collagen, such as collagenI, collagen II, collagen III, collagen IV, collagen V, collagen VI,collagen VII, collagen VIII, collagen IX, and collagen X. The matrix mayfurther comprise elastin. The matrix further comprises a syntheticpolymer such as poly(lactide-co-glycolides) (PLGA). Thus, theelectrospun matrix can comprise at least one natural component and atleast one synthetic polymer component.

In another aspect, the invention pertains to a method of controlling therelease of a therapeutic agent at a target site. The method involvesproviding a matrix having a three-dimensional ultrastructure ofinterconnected fibers and pores that permit cell attachment. The matrixalso comprises nanoparticles, such as quantum dots, incorporated withinor on a the matrix, and these nanoparticles can be coupled to atherapeutic agent. This matrix can be delivered to a target site, andthe therapeutic agent released at the target site to provide acontrolled release of the therapeutic agent. The matrix can be selectedfrom the group consisting of an electrospun matrix, a decellularizedmatrix, and a synthetic polymer matrix.

If the matrix is an electropsun matrix, the nanoparticles can beincorporated within the matrix during electrospining of the electrospunmatrix, or applied to the surface of the matrix after eletcrospinning byreacting the surface of the matrix with the nanoparticles. If the matrixis a decellularized matrix or a synthetic polymer matrix, thenanoparticles can be incorporated on the matrix by reacting the surfaceof the matrix with the nanoparticles.

In other aspects or the invention, scaffolds for a variety of purposesare described using electrospin matrices including, but not limited toblood vessels, heart valves, and vascular and cardiac structuresgenerally.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of an electrospin apparatus;

FIG. 2 is a schematic of the conjugation of heparin on quantum dots;

FIG. 3 is an electrospin nanofiber;

FIG. 4 is graph of pressure-diameter curves of vascular graft scaffolds;

FIG. 5A is a graph of axial and circumferential stress-strain data fromuniaxial testing of two decellularized constructs;

FIG. 5B is a graph of axial and circumferential stress-strain data fromuniaxial testing of an electrospun vessel;

FIG. 6A is a graph of cell viability of endothelial cells cultured onfour matrices;

FIG. 6B is a graph of mitochondrial metabolic activity of endothelialcells cultured on four matrices;

FIG. 7A is a graph of cell viability of endothelial cells cultured onfive matrices;

FIG. 7B is a graph of mitochondrial metabolic activity of endothelialcells cultured on five matrices;

FIG. 8 is a graph of microcapsules containing heparin which show heparinrelease upon near infra-red irradiation; and

FIG. 9 is a graph showing the quantification of remaining heparin fromretrieved vascular scaffolds.

DETAILED DESCRIPTION

So that the invention may more readily be understood, certain terms arefirst defined:

The term “attach” or “attaches” as used herein refers to cells thatadhere directly or indirectly to a substrate as well as to cells thatadhere to other cells.

The phrase “biocompatible substrate” as used herein refers to a materialthat is suitable for implantation into a subject onto which a cellpopulation can be deposited. A biocompatible substrate does not causetoxic or injurious effects once implanted in the subject. In oneembodiment, the biocompatible substrate is a polymer with a surface thatcan be shaped into the desired structure that requires repairing orreplacing. The polymer can also be shaped into a part of an structurethat requires repairing or replacing. The biocompatible substrateprovides the supportive framework that allows cells to attach to it, andgrow on it. Cultured populations of cells can then be grown on thebiocompatible substrate, which provides the appropriate interstitialdistances required for cell-cell interaction.

The term “subject” as used herein is intended to include livingorganisms in which an immune response is elicited. Preferred subjectsare mammals. Examples of subjects include but are not limited to,humans, monkeys, dogs, cats, mice, rates, cows, horses, pigs, goats andsheep.

The term “decellularized” or “decellularization” as used herein refersto a biostructure (e.g., an organ, or part of an organ), from which thecellular and tissue content has been removed leaving behind an intactacellular infra-structure. Organs such as the kidney are composed ofvarious specialized tissues. The specialized tissue structures of anorgan, or parenchyma, provide the specific function associated with theorgan. The supporting fibrous network of the organ is the stroma. Mostorgans have a stromal framework composed of unspecialized connectingtissue which supports the specialized tissue. The process ofdecellularization removes the specialized tissue, leaving behind thecomplex three-dimensional network of connective tissue. The connectivetissue infra-structure is primarily composed of collagen. Thedecellularized structure provides a biocompatible substrate onto whichdifferent cell populations can be infused. Decellularized biostructurescan be rigid, or semi-rigid, having an ability to alter their shapes.Examples of decellularized organs useful in the present inventioninclude, but are not limited to, the heart, kidney, liver, pancreas,spleen, bladder, ureter and urethra.

The phrase “three-dimensional scaffold” as used herein refers to theresidual infra-structure formed when a natural biostructure, e.g. anorgan, is decellularized. This complex, three-dimensional, scaffoldprovides the supportive framework that allows cells to attach to it, andgrow on it. Cultured populations of cells can then be grown on thethree-dimensional scaffold, which provides the exact interstitialdistances required for cell-cell interaction. This provides areconstructed organ that resembles the native in vivo organ. Thisthree-dimensional scaffold can be perfused with a population of culturedcells, e.g., endothelial cells, which grow and develop to provide anendothelial tissue layer capable of supporting growth and development ofat least one additional cultured cell population.

The term “natural biostructure” as used herein refers to a biologicalarrangement found within a subject, for example, organs, that includebut are not limited, heart, kidney, liver, pancreas, spleen, bladder,ureter and urethra. The term “natural biostructure” is also intended toinclude parts of biostructures, for example parts of organs, forexample, the renal artery of a kidney.

The terms “electrospinning” or “electrospun,” as used herein refers toany method where materials are streamed, sprayed, sputtered, dripped, orotherwise transported in the presence of an electric field. Theelectrospun material can be deposited from the direction of a chargedcontainer towards a grounded target, or from a grounded container in thedirection of a charged target. In particular, the term “electrospinning”means a process in which fibers are formed from a charged solutioncomprising at least one natural biological material, at least onesynthetic polymer material, or a combination thereof by streaming theelectrically charged solution through an opening or orifice towards agrounded target.

A natural biological material can be a naturally occurring organicmaterial including any material naturally found in the body of a mammal,plant, or other organism. A synthetic polymer material can be anymaterial prepared through a method of artificial synthesis, processing,or manufacture. Preferably the synthetic materials is a biologicallycompatible material. The natural or synthetic materials are also thosethat are capable of being charged under an electric field.

The terms “solution” and “fluid” as used herein describe a liquid thatis capable of being charged and which comprises at least one naturalmaterial, at least one synthetic material, or a combination thereof. Ina preferred embodiment, the fluid comprises at least one type ofcollagen, an additional natural material such as at least one type ofelastin and at least one synthetic polymer, e.g., poly-L glycolic acid(PLGA).

The term “co-polymer” as used herein is intended to encompassco-polymers, ter-polymers, and higher order multiple polymercompositions formed by block, graph or random combination of polymericcomponents.

The terms “nanoparticles,” “nanostructures,” and “quantum dots” are usedinterchangeably herein to describe materials having dimensions of theorder of one or a few nanometers to a few micrometers, more preferablyfrom about 1 to about 1000 nanometers.

I Electrospun Matrices

The invention pertains to methods and compositions for producing andusing electrospun matrices. The process of electrospinning generallyinvolves the creation of an electrical field at the surface of a liquid.The resulting electrical forces create a jet of liquid which carrieselectrical charge. The liquid jets may be attracted to otherelectrically charged objects at a suitable electrical potential. As thejet of liquid elongates and travels, it will harden and dry. Thehardening and drying of the elongated jet of liquid may be caused bycooling of the liquid, i.e., where the liquid is normally a solid atroom temperature; evaporation of a solvent, e.g., by dehydration,(physically induced hardening); or by a curing mechanism (chemicallyinduced hardening). The produced fibers are collected on a suitablylocated, oppositely charged target substrate.

The electrospinning apparatus includes an electrodepositing mechanismand a target substrate. The electrodepositing mechanism includes atleast one container to hold the solution that is to be electrospun. Thecontainer has at least one orifice or nozzle to allow the streaming ofthe solution from the container. If there are multiple containers, aplurality of nozzles may be used.

One or more pumps (e.g., a syringe pump) used in connection with thecontainer can be used to control the flow of solution streaming from thecontainer through the nozzle. The pump can be programmed to increase ordecrease the flow at different points during electrospinning.

The electrospinning occurs due to the presence of a charge in either theorifices or the target, while the other is grounded. In someembodiments, the nozzle or orifice is charged and the target isgrounded. Those of skill in the electrospinning arts will recognize thatthe nozzle and solution can be grounded and the target can beelectrically charged.

The target can also be specifically charged or grounded along apreselected pattern so that the solution streamed from the orifice isdirected into specific directions. The electric field can be controlledby a microprocessor to create an electrospun matrix having a desiredgeometry. The target and the nozzle or nozzles can be engineered to bemovable with respect to each other thereby allowing additional controlover the geometry of the electrospun matrix to be formed. The entireprocess can be controlled by a microprocessor that is programmed withspecific parameters that will obtain a specific preselected electrospunmatrix.

In embodiments in which two materials combine to form a third material,the solutions containing these components can be mixed togetherimmediately before they are streamed from an orifice in theelectrospinning procedure. In this way, the third material formsliterally as the microfibers in the electrospinning process.

While the following is a description of a preferred method, otherprotocols can be followed to achieve the same result. In FIG. 1, acontainer 10, (e.g., a syringe or micropipette), with an orifice ornozzle 12 (e.g. a Taylor cone), is filled with a solution with at leastone natural material, and at least one synthetic material 14. Thecontainer 10 is suspended opposite a grounded target 16, such as a metalground screen. A fine wire 18 is placed in the solution to charge thesolution in the container to a high voltage. At a specific voltagedetermined for each solution, the solution in the container nozzle isdirected towards the grounded target. The single jet stream 20 ofmaterials forms a splayed jet 22, upon reaching the grounded target,e.g., a rapidly rotating mandrel. The splayed jet collects and dries toform a three-dimensional, ultra thin, interconnected matrix ofelectrospun fibers. In some embodiments, a plurality of containers canbe used with each of the containers holding a different compound.

Minimal electrical current is involved in the electrospinning process,therefore the process does not denature the materials that form theelectrospun matrix, because the current causes little or no temperatureincrease in the solutions during the procedure.

The electrospinning process can be manipulated to meet the specificrequirements for any given application of the electrospun matrix. In oneembodiment, a syringe can be mounted on a frame that moves in the x, yand z planes with respect to the grounded substrate. In anotherembodiment, a syringe can be mounted around a grounded substrate, forinstance a tubular mandrel. In this way, the materials that form thematrix streamed from the a syringe can be specifically aimed orpatterned. Although the micropipette can be moved manually, the frameonto which the a syringe is mounted can also be controlled by amicroprocessor and a motor that allows the pattern of streaming to bepredetermined. Such microprocessors and motors are known to one ofordinary skill in the art, for example matrix fibers can be oriented ina specific direction, they can be layered, or they can be programmed tobe completely random and not oriented.

The degree of branching can be varied by many factors including, but notlimited to, voltage (for example ranging from about 0 to 30,000 volts),distance from a syringe tip to the substrate (for example from 1-100 cm,0-40 cm, and 1-10 cm), the speed of rotation, the shape of the mandrel,the relative position of the a syringe tip and target (i.e. in front of,above, below, aside etc.), and the diameter of a syringe tip(approximately 0-2 mm), and the concentration and ratios of compoundsthat form the electrospun matrix. Other parameters which are importantinclude those affecting evaporation of solvents such as temperature,pressure, humidity. The molecular weight of the polymer improves itsability to entangle and form fibers, and polymers with the molecularweight of 100 kDa generally performed. Those skilled in the art willrecognize that these and other parameters can be varied to formelectrospun materials with characteristics that are particularly adaptedfor specific applications.

The geometry of the grounded target can be modified to produce a desiredmatrix. By varying the ground geometry, for instance having a planar orlinear or multiple points ground, the direction of the streamingmaterials can be varied and customized to a particular application. Forinstance, a grounded target comprising a series of parallel lines can beused to orient electrospun materials in a specific direction. Thegrounded target can be a cylindrical mandrel whereby a tubular matrix isformed. The ground can be variable surface that can be controlled by amicroprocessor that dictates a specific ground geometry that isprogrammed into it. Alternatively, the ground can be mounted on a framethat moves in the x, y, and z planes with respect to a stationarycontainer, e.g., a syringe or micropipette tip.

Electrospinning allows great flexibility and allows for customizing theconstruct to virtually any shape needed. In shaping matrices, portionsof the matrix may be sealed to one another by, for example, heatsealing, chemical sealing, and application of mechanical pressure or acombination thereof. The electrospun compositions may be shaped intoshapes such as a skin patch, an intraperitoneal implant, a subdermalimplant, the interior lining of a stent, a cardiovascular valve, atendon, a ligament, a muscle implant, a nerve guide and the like.

The electrospinning process can also be modified for example by (i)using mixed solutions (for example, materials along with substances suchas cells, growth factors, or both) in the same container to producefibers composed of electrospun compounds as well as one or moresubstances to produce a “blend,” and (ii) applying agents such as Teflononto the target to facilitate the removal of electrospun compounds fromthe target (i.e. make the matrix more slippery so that the electrospunmatrix does not stick to the target).

The various properties of the electrospun materials can be adjusted inaccordance with the needs and specifications of the cells to besuspended and grown within them. The porosity, for instance, can bevaried in accordance with the method of making the electrospun materialsor matrix. Electrospinning a particular matrix, for instance, can bevaried by fiber size and density. If the cells to be grown in the matrixrequire a high nutrient flow and waste expulsion, then a loose matrixcan be created. On the other hand, if the tissue to be made requires adense environment, then a dense matrix can be designed. Porosity can bemanipulated by mixing salts or other extractable agents. Removing thesalt will leave holes of defined sizes in the matrix.

One embodiment for appropriate conditions for electrospinning a matrixis as follows. For electrospinning a matrix by combining 45% collagen I,15% elastin and 40% PLGA, the appropriate approximate ranges are:voltage 0-30,000 volts (10-100 kV potential preferably 15-30 kV); pH 7.0to 8.0; temperature 20 to 40° C., e.g., room temperature of 25° C.; andthe distance from the container to the grounded plate can range fromabout 1 cm to about 100 cm, preferably about 1 cm to 10 cm. In additionto depositing the charged fibers on the grounded plate, the fibers canbe deposited onto another substrate such as a stainless steel mandrel.The mandrel can be rotated at 20-1000 rpm, preferably about 300-700 rpm.

Examples of naturally occurring materials include, but are not limitedto, amino acids, peptides, denatured peptides such as gelatin fromdenatured collagen, polypeptides, proteins, carbohydrates, lipids,nucleic acids, glycoproteins, lipoproteins, glycolipids,glycosaminoglycans, and proteoglycans. In a preferred embodiment, thematerials compound is an extracellular matrix material, including butnot limited to collagen, fibrin, elastin, laminin, fibronectin,hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatansulfate, heparin sulfate, heparin, and keratan sulfate, andproteoglycans. These materials may be isolated from humans or otheranimals or cells. A preferred natural compound is collagen. Examples ofcollagen include, but are not limited to collagen I, collagen II,collagen III, collagen IV, collagen V, collagen VI, collagen VII,collagen VIII, collagen IX, and collagen X. Another preferred naturalcompound is elastin. Elastin fibers are responsible for the elasticproperties of several tissues. Elastin is found, for example, in skin,blood vessels, and tissues of the lung where it imparts strength,elasticity and flexibility.

One class of synthetic polymer materials are biocompatible syntheticpolymers. Such polymers include, but are not limited to,poly(urethanes), poly(siloxanes) or silicones, poly(ethylene),poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate),poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinylalcohol) (PVA), poly(acrylic acid), poly(vinyl acetate), polyacrylamide,poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylicacid), polylactic acid (PLA), polyglycolic acids (PGA),poly(lactide-co-glycolides) (PLGA), nylons, polyamides, polyanhydrides,poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinylacetate), polyvinylhydroxide, poly(ethylene oxide) (PEO) andpolyorthoesters or any other similar synthetic polymers that may bedeveloped that are biologically compatible. A preferred syntheticpolymer is PGLA.

In matrices composed of electrospun elastin (for elasticity),electrospun collagen (to promote cell infiltration and lend mechanicalintegrity), and other components, such as PLGA, PGA, PLA, PEO, PVA, orother blends, the relative ratio of the different components in thematrix can be tailored to specific applications (e.g. more elastin, lesscollagen depending on the tissue to be engineered).

Electrospun matrices can be formed of electrospun fibers of syntheticpolymers that are biologically compatible. The term “biologicallycompatible” includes copolymers and blends, and any other combinationsof the forgoing either together or with other polymers. The use of thesepolymers will depend on given applications and specifications required.A more detailed discussion of these polymers and types of polymers isset forth in Brannon-Peppas, Lisa, “Polymers in Controlled DrugDelivery,” Medical Plastics and Biomaterials, November 1997, which isincorporated herein by reference.

When both natural and synthetic materials are used in an electrospunmatrix, the natural material component can range from about 5 percent toabout 95 percent, preferably from about 25 percent to about 75 percentby weight. The synthetic material component can range from about 5percent to about 95 percent, preferably from about 25 percent to about75 percent by weight. In certain embodiments, both collagen and elastincan be included as natural material components, preferably with apredominance of collagen, e.g., greater than 40 percent of the naturalmaterial component. Ratios of collagen, elastin, and PLGA may betailored to fit the application: for instances, normal levels ofcollagen and elastin vary from the more elastic vessels closer to theheart to less compliant vessels further from the heart. A vessel such asthe aorta would have greater elastin content than a distal vessel. Thepercentages of collagen I, elastin, and other collagens (collagen IIIfor blood vessels or collagen II, for instance, for cartilage) may bewhatever is desired, as long as the molecular weight of these collagensis sufficient to form fibers in the electrospinning process. Ratios ofcollagen I may be from 40% to 80%, or 50%-100%. Elastin may also be usedin higher ratios from 5% to 50%. PLGA or another synthetic biodegradablepolymer may be used as desired in ratios from 5 to 80%. For a completelybiological substrate, synthetic polymers may be omitted completely andonly biological polymers may be used.

The compounds to be electrospun can be present in the solution at anyconcentration that will allow electrospinning. In one embodiment, thecompounds may be electrospun are present in the solution atconcentrations between 0 and about 1.000 g/ml. In another embodiment,the compounds to be electrospun are present in the solution at totalsolution concentrations between 10-15 w/v % (100-150 mg/ml or 0-0.1g/L).

The compounds can be dissolved in any solvent that allows delivery ofthe compound to the orifice, tip of a syringe, under conditions that thecompound is electrospun. Solvents useful for dissolving or suspending amaterial or a substance will depend on the compound. Electrospinningtechniques often require more specific solvent conditions. For example,collagen can be electrodeposited as a solution or suspension in water,2,2,2-trifluoroethanol, 1,1,1,3,3,3-hexafluoro-2-propanol (also known ashexafluoroisopropanol or HFIP), or combinations thereof. Fibrin monomercan be electrodeposited or electrospun from solvents such as urea,monochloroacetic acid, water, 2,2,2-trifluoroethanol, HFIP, orcombinations thereof. Elastin can be electrodeposited as a solution orsuspension in water, 2,2,2-trifluoroethanol, isopropanol, HFIP, orcombinations thereof, such as isopropanol and water. In one desirableembodiment, elastin is electrospun from a solution of 70% isopropanoland 30% water containing 250 mg/ml of elastin. Other lower orderalcohols, especially halogenated alcohols, may be used. Other solventsthat may be used or combined with other solvents in electrospinningnatural matrix materials include acetamide, N-methylformamide,N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO),dimethylacetamide, N-methylpyrrolidone (NMP), acetic acid,trifluoroacetic acid, ethyl acetate, acetonitrile, trifluoroaceticanhydride, 1,1,1-trifluoroacetone, maleic acid, hexafluoroacetone.Organic solvents such as methanol, chloroform, and trifluoroethanol(TFE) and emulsifying agents.

The selection of a solvent is based in part on consideration ofsecondary forces that stabilize polymer-polymer interactions and thesolvent's ability to replace these with strong polymer-solventinteractions. In the case of polypeptides such as collagen, and in theabsence of covalent crosslinking, the principal secondary forces betweenchains are: (1) coulombic, resulting from attraction of fixed charges onthe backbone and dictated by the primary structure (e.g., lysine andarginine residues will be positively charged at physiological pH, whileaspartic or glutamic acid residues will be negatively charged); (2)dipole-dipole, resulting from interactions of permanent dipoles; thehydrogen bond, commonly found in polypeptides, is the strongest of suchinteractions; and (3) hydrophobic interactions, resulting fromassociation of non-polar regions of the polypeptide due to a lowtendency of non-polar species to interact favorably with polar watermolecules. Therefore, solvents or solvent combinations that canfavorably compete for these interactions can dissolve or dispersepolypeptides. For example, HFIP and TFE possess a highly polar OH bondadjacent to a very hydrophobic fluorinated region. While not wanting tobe bound by the following theories, it is believed that the alcoholportion can hydrogen bond with peptides, and can also solvate charges onthe backbone, thus reducing Coulombic interactions between molecules.Additionally, the hydrophobic portions of these solvents can interactwith hydrophobic domains in polypeptides, helping to resist the tendencyof the latter to aggregate via hydrophobic interactions. It is furtherbelieved that solvents such as HFIP and TFE, due to their lower overallpolarities compared to water, do not compete well for intramolecularhydrogen bonds that stabilize secondary structures such as an alphahelix. Consequently, alpha helices in these solvents are believed to bestabilized by virtue of stronger intramolecular hydrogen bonds. Thestabilization of polypeptide secondary structures in these solvents isbelieved desirable, especially in the cases of collagen and elastin, topreserve the proper formation of collagen fibrils duringelectrospinning.

In one embodiment, the solvent has a relatively high vapor pressure topromote the stabilization of an electrospinning jet to create a fiber asthe solvent evaporates. In embodiments involving higher boiling pointsolvents, it is often desirable to facilitate solvent evaporation bywarming the spinning or spraying solution, and optionally theelectrospinning stream itself, or by electrospinning in reducedatmospheric pressure. It is also believed that creation of a stable jetresulting in a fiber is facilitated by a high surface tension of thepolymer/solvent mixture.

Similar to conventional electrospinning, midair electrospinning can beused which employs the same experimental set-up as other electrospinningtechniques. However, in order to precipitate fibers before they reachthe grounded target, the distance from the needle to the grounded targetcan be increased. For example, increasing the distance from the 10-30 cmto a distance of 30-40 cm. The field strength can be maintained oraltered by increasing the applied potential at the needle tip.Increasing the distance from the needle tip to the grounded targetallows the polymer jet to experience a longer “flight time.” The addedflight time, allows the solvent to be completely evaporated from the jetallowing the fibers to fully develop.

By varying the composition of the fibers being electrospun, it will beappreciated that fibers having different physical or chemical propertiesmay be obtained. This can be accomplished either by spinning a liquidcontaining a plurality of components, each of which may contribute adesired characteristic to the finished product, or by simultaneouslyspinning fibers of different compositions from multiple liquid sources,that are then simultaneously deposited to form a matrix. The resultingmatrix comprises layers of intermingled fibers of different compounds.This plurality of layers of different materials can convey a desiredcharacteristic to the resulting composite matrix with each differentlayer providing a different property, for example one layer maycontribute to elasticity while another layer contributes to themechanical strength of the composite matrix. These methods can be usedto create tissues with multiple layers such as blood vessels.

The electrospun matrix has an ultrastructure with a three-dimensionalnetwork that supports cell growth, proliferation, differentiation anddevelopment. The spatial distance between the fibers plays an importantrole in cells being able to obtain nutrients for growth as well as forallowing cell-cell interactions to occur. Thus, in various embodimentsof the invention, the distance between the fibers may be about 50nanometers, about 100 nanometers, about 150 nanometers, about 200nanometers, about 250 nanometers, about 300 nanometers, about 350nanometers, about 600 nanometers, about 750 nanometers, about 800nanometers, about 850 nanometers, about 900 nanometers, about 950nanometers, about 1000 nanometers (1 micron), 10 microns, 10 microns, 50microns, about 100 microns, about 150 microns, about 200 microns, about250 microns, about 300 microns, about 350 microns, about 400 microns,about 450 microns, or about 500 microns. In various embodiments thedistance between the fibers may be less than 50 nanometers or greaterthan 500 microns and any length between the quoted ranges as well asintegers.

Additionally, in various embodiments of the invention, the fibers canhave a diameter of about 50 nanometers, about 100 nanometers, about 150nanometers, about 200 nanometers, about 250 nanometers, about 300nanometers, about 350 nanometers, about 600 nanometers, about 750nanometers, about 800 nanometers, about 850 nanometers, about 900nanometers, about 950 nanometers, about 1000 nanometers (1 micron), 50microns, about 100 microns, about 150 microns, about 200 microns, about250 microns, about 300 microns, about 350 microns, about 400 microns,about 450 microns, or about 500 microns, or the diameter may be lessthan 50 nanometers or greater than 500 microns and any diameter betweenthe quoted ranges as well as integers.

The pore size in an electrospun matrix can also be controlled throughmanipulation of the composition of the material and the parameters ofelectrospinning. In some embodiments, the electrospun matrix has a poresize that is small enough to be impermeable to one or more types ofcells. In one embodiment, the average pore diameter is about 500nanometers or less. In another embodiment, the average pore diameter isabout 1 micron or less. In another embodiment, the average pore diameteris about 2 microns or less. In another embodiment, the average porediameter is about 5 microns or less. In another embodiment, the averagepore diameter is about 8 microns or less. Some embodiments have poresizes that do not impede cell infiltration. In another embodiment, thematrix has a pore size between about 0.1 and about 100 μm². In anotherembodiment, the matrix has a pore size between about 0.1 and about 50μm². In another embodiment, the matrix has a pore size between about 1.0μm and about 25 μ□m. In another embodiment, the matrix has a pore sizebetween about 1.0 μm and about 5 μm. Infiltration can also beaccomplished with implants with smaller pore sizes. The pore size of anelectrospun matrix can be readily manipulated through control of processparameters, for example by controlling fiber deposition rate throughelectric field strength and mandrel motion, by varying solutionconcentration (and thus fiber size). Porosity can also be manipulated bymixing porogenic materials, such as salts or other extractable agents,the dissolution of which will leave holes of defined sizes in thematrix. The pore size can also be controlled by the amount ofcross-linking present in the matrix.

The mechanical properties of the matrix will depend on the polymermolecular weight and polymer type/mixture. It will also depend onorientation of the fibers (preferential orientation can be obtained bychanging speed of a rotating or translating surface during the fibercollection process), fiber diameter and entanglement. The cross-linkingof the polymer will also effect its mechanical strength after thefabrication process.

The electrospun matrix can be cross linked to increase its stability andstrength. The crosslinking can generally be conducted at roomtemperature and neutral pH conditions, however, the conditions may bevaried to optimize the particular application and crosslinking chemistryutilized. For crosslinking using the EDC chemistry with NHS in MES/EtOH,pH of from 4.0 to 8.0 and temperatures from 0° C. to room temperature(25° C.) for two hours, can be used. It is known that highertemperatures are unpreferred for this chemistry due to decomposition ofEDC. Similarly, basic pH (e.g., 8-14) is also unpreferred for thisreason when using this chemistry. Other crosslinking chemistries canalso be used for example, by soaking the electrospun matrix in 20%dextran solution (to reduce shrinking), followed by 1% glutaraldehydesolution. Yet other cross-linking chemistries involve usingpoly(ethylene glycol) (PEG) as a spacer in a crosslinking agent with anN-protected amino acid.

II. Decellularized Matrices

Natural biostructures, e.g. an organ, can be obtained from a donor ofthe same species as the subject, for example, a human cadaver kidney fora human kidney recipient. The natural biostructure can also be obtainedfrom a different species which includes, but is not limited to, monkeys,dogs, cats, mice, rats, cows, horses, pigs, goats and sheep. The naturalbiostructure can also be obtained from the subject requiring areconstructed organ, for example, a subject with one dysfunctionalkidney and one functional kidney, can have the dysfunctional kidneyremoved and decellularized using the process described below. Thedecellularized kidney of the subject can be used as thethree-dimensional scaffold to reconstruct an artificial kidney usingcultured endothelial cells and kidney cells isolated from the subject.The artificial reconstructed kidney can be implanted back into thesubject for further development.

Biostructures, e.g., whole organs, or parts of organs can bedecellularized by removing the entire cellular and tissue content fromthe organ. The decellularization process comprises a series ofsequential extractions. One key feature of this extraction process isthat harsh extraction that may disturb or destroy the complexinfra-structure of the biostructure, be avoided. The first step involvesremoval of cellular debris and solubilization of the cell membrane. Thisis followed by solubilization of the nuclear cytoplasmic components anthe nuclear components.

Preferably, the biostructure, e.g., an organ, is decellularized byremoving the cell membrane and cellular debris surrounding the organusing gentle mechanical disruption methods. The gentle mechanicaldisruption methods must be sufficient to disrupt the cellular membrane.However, the process of decellularization should avoid damage ordisturbance of the biostructure's complex infra-structure. Gentlemechanical disruption methods include scraping the surface of the organ,agitating the organ, or stirring the organ in a suitable volume offluid, e.g., distilled water. In one preferred embodiment, the gentlemechanical disruption method includes magnetically stirring (e.g., usinga magnetic stir bar and a magnetic plate) the organ in a suitable volumeof distilled water until the cell membrane is disrupted and the cellulardebris has been removed from the organ.

After the cell membrane has been removed, the nuclear and cytoplasmiccomponents of the biostructure are removed. This can be performed bysolubilizing the cellular and nuclear components without disrupting theinfra-structure. To solubilize the nuclear components, non-ionicdetergents or surfactants may be used. Examples of non-ionic detergentsor surfactants include, but are not limited to, the Triton series,available from Rohm and Haas of Philadelphia, Pa., which includes TritonX-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, andTriton DF-16, available commercially from many vendors; the Tweenseries, such as monolaurate (Tween 20), monopalmitate (Tween 40),monooleate (Tween 80), and polyoxethylene-23-lauryl ether (Brij. 35),polyoxyethylene ether W-1 (Polyox), and the like, sodium cholate,deoxycholates, CHAPS, saponin, n-Decyl β-D-glucopuranoside, n-heptyl β-Dglucopyranoside, n-Octyl-α-D-glucopyranoside and Nonidet P-40.

One skilled in the art will appreciate that a description of compoundsbelonging to the foregoing classifications, and vendors may becommercially obtained and may be found in “Chemical Classification,Emulsifiers and Detergents”, McCutcheon's, Emulsifiers and Detergents,1986, North American and International Editions, McCutcheon Division, MCPublishing Co., Glen Rock, N.J., U.S.A. and Judith Neugebauer, A Guideto the Properties and Uses of Detergents in Biology and Biochemistry,Calbiochem, Hoechst Celanese Corp., 1987. In one preferred embodiment,the non-ionic surfactant is the Triton series, preferably, Triton X-100.

The concentration of the non-ionic detergent may be altered depending onthe type of biostructure being decellularized. For example, for delicatetissues, e.g., blood vessels, the concentration of the detergent shouldbe decreased. Preferred concentrations ranges non-ionic detergent can befrom about 0.001 to about 2.0% (w/v). More preferably, about 0.05 toabout 1.0% (w/v). Even more preferably, about, 0.1% (w/v) to about 0.8%(w/v). Preferred concentrations of these range from about 0.001 to about0.2% (w/v), with about 0.05 to about 0.1% (w/v) particular preferred.

The cytoskeletal component, comprising consisting of the densecytoplasmic filament networks, intercellular complexes and apicalmicrocellular structures, may be solubilized using alkaline solution,such as, ammonium hydroxide. Other alkaline solution consisting ofammonium salts or their derivatives may also be used to solubilize thecytoskeletal components. Examples of other suitable ammonium solutionsinclude ammonium sulphate, ammonium acetate and ammonium hydroxide. In apreferred embodiment, ammonium hydroxide is used.

The concentration of the alkaline solutions, e.g., ammonium hydroxide,may be altered depending on the type of biostructure beingdecellularized. For example, for delicate tissues, e.g., blood vessels,the concentration of the detergent should be decreased. Preferredconcentrations ranges can be from about 0.001 to about 2.0% (w/v). Morepreferably, about 0.005 to about 0.1% (w/v). Even more preferably,about, 0.01% (w/v) to about 0.08% (w/v).

The decellularized, lyophilized structure may be stored at a suitabletemperature until required for use. Prior to use, the decellularizedstructure can be equilibrated in suitable isotonic buffer or cellculture medium. Suitable buffers include, but are not limited to,phosphate buffered saline (PBS), saline, MOPS, HEPES, Hank's BalancedSalt Solution, and the like. Suitable cell culture medium includes, butis not limited to, RPMI 1640, Fisher's, Iscove's, McCoy's, Dulbecco'smedium, and the like.

III. Synthetic Matrices

The invention also pertains to generating artificial tissue constructsby seeding cultured tissue cells onto or into available biocompatiblematrices. Biocompatible refers to materials that do not have toxic orinjurious effects on biological functions. Biodegradable refers tomaterial that can be absorbed or degraded in a patient's body.Representative materials for forming the biodegradable material includenatural or synthetic polymers, such as, collagen, poly(alpha esters)such as poly(lactate acid), poly(glycolic acid), polyorthoesters amdpolyanhydrides and their copolymers, which degraded by hydrolysis at acontrolled rate and are reabsorbed. These materials provide the maximumcontrol of degradability, manageability, size and configuration.Preferred biodegradable polymer materials include polyglycolic acid andpolyglactin, developed as absorbable synthetic suture material.

Polyglycolic acid and polyglactin fibers may be used as supplied by themanufacturer. Other biodegradable materials include, but are not limitedto, cellulose ether, cellulose, cellulosic ester, fluorinatedpolyethylene, phenolic, poly-4-methylpentene, polyacrylonitrile,polyamide, polyamideimide, polyacrylate, polybenzoxazole, polycarbonate,polycyanoarylether, polyester, polyestercarbonate, polyether,polyetheretherketone, polyetherimide, polyetherketone, polyethersulfone,polyethylene, polyfluoroolefin, polyimide, polyolefin, polyoxadiazole,polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene,polysulfide, polysulfone, polytetrafluoroethylene, polythioether,polytriazole, polyurethane, polyvinyl, polyvinylidene fluoride,regenerated cellulose, silicone, urea-formaldehyde, or copolymers orphysical blends of these materials. The material may be impregnated withsuitable antimicrobial agents and may be colored by a color additive toimprove visibility and to aid in surgical procedures.

In some embodiments, attachment of the cells to the biocompatiblesubstrate is enhanced by coating the matrix with compounds such asbasement membrane components, agar, agarose, gelatin, gum arabic,collagens, fibronectin, laminin, glycosaminoglycans, mixtures thereof,and other materials having properties similar to biological matrixmolecules known to those skilled in the art of cell culture. Mechanicaland biochemical parameters ensure the matrix provide adequate supportfor the cells with subsequent growth and proliferation. Factors,including nutrients, growth factors, inducers of differentiation ordedifferentiation, products of secretion, immunomodulators, inhibitorsof inflammation, regression factors, biologically active compounds whichenhance or allow ingrowth of the lymphatic network or nerve fibers, anddrugs, can be incorporated into the matrix or provided in conjunctionwith the matrix. Similarly, polymers containing peptides such as theattachment peptide RGD (Arg-Gly-Asp) can be synthesized for use informing matrices.

Coating refers to coating or permeating a matrix with a material suchas, liquefied copolymers (poly-DL-lactide co-glycolide 50:50 80 mg/mlmethylene chloride) to alter its mechanical properties. Coating may beperformed in one layer, or multiple layers until the desired mechanicalproperties are achieved. These shaping techniques may be employed incombination, for example, a polymeric matrix can be weaved, compressionmolded and glued together. Furthermore different polymeric materialsshaped by different processes may be joined together to form a compositeshape. The composite shape can be a laminar structure. For example, apolymeric matrix may be attached to one or more polymeric matrixes toform a multilayer polymeric matrix structure. The attachment may beperformed by any suitable means such as gluing with a liquid polymer,stapling, suturing, or a combination of these methods. In addition, thepolymeric matrix may be formed as a solid block and shaped by laser orother standard machining techniques to its desired final form. Lasershaping refers to the process of removing materials using a laser.

The polymers can be characterized for mechanical properties such astensile strength using an Instron tester, for polymer molecular weightby gel permeation chromatography (GPC), glass transition temperature bydifferential scanning calorimetry (DSC) and bond structure by infrared(IR) spectroscopy; with respect to toxicology by initial screening testsinvolving Ames assays and in vitro teratogenicity assays, andimplantation studies in animals for immunogenicity, inflammation,release and degradation studies. In vitro cell attachment and viabilitycan be assessed using scanning electron microscopy, histology, andquantitative assessment with radioisotopes.

Substrates can be treated with additives or drugs prior to implantation(before or after the polymeric substrate is seeded with cells), e.g., topromote the formation of new tissue after implantation. Thus, forexample, growth factors, cytokines, extracellular matrix components, andother bioactive materials can be added to the substrate to promote grafthealing and formation of new tissue. Such additives will in general beselected according to the tissue or organ being reconstructed oraugmented, to ensure that appropriate new tissue is formed in theengrafted organ or tissue (for examples of such additives for use inpromoting bone healing, see, e.g., Kirker-Head, C. A. Vet. Surg. 24 (5):408-19 (1995)). For example, vascular endothelial growth factor (VEGF,see, e.g., U.S. Pat. No. 5,654,273 herein incorporated by reference) canbe employed to promote the formation of new vascular tissue. Growthfactors and other additives (e.g., epidermal growth factor (EGF),heparin-binding epidermal-like growth factor (HBGF), fibroblast growthfactor (FGF), cytokines, genes, proteins, and the like) can be added inamounts in excess of any amount of such growth factors (if any) whichmay be produced by the cells seeded on the substrate. Such additives arepreferably provided in an amount sufficient to promote the formation ofnew tissue of a type appropriate to the tissue or organ, which is to berepaired or augmented (e.g., by causing or accelerating infiltration ofhost cells into the graft). Other useful additives include antibacterialagents such as antibiotics.

The biocompatible substrate may be shaped using methods such as, solventcasting, compression molding, filament drawing, meshing, leaching,weaving and coating. In solvent casting, a solution of one or morepolymers in an appropriate solvent, such as methylene chloride, is castas a branching pattern relief structure. After solvent evaporation, athin film is obtained. In compression molding, the substrate is pressedat pressures up to 30,000 pounds per square inch into an appropriatepattern. Filament drawing involves drawing from the molten polymer andmeshing involves forming a mesh by compressing fibers into a felt-likematerial. In leaching, a solution containing two materials is spreadinto a shape close to the final form of the tissue. Next a solvent isused to dissolve away one of the components, resulting in poreformation. (See Mikos, U.S. Pat. No. 5,514,378, hereby incorporated byreference).

In nucleation, thin films in the shape of the tissue are exposed toradioactive fission products that create tracks of radiation damagedmaterial. Next, the polycarbonate sheets are etched with acid or base,turning the tracks of radiation-damaged material into pores. Finally, alaser may be used to shape and burn individual holes through manymaterials to form an tissue structure with uniform pore sizes. Thesubstrate can be fabricated to have a controlled pore structure thatallows nutrients from the culture medium to reach the deposited cellpopulation. In vitro cell attachment and cell viability can be assessedusing scanning electron microscopy, histology and quantitativeassessment with radioisotopes.

Thus, the substrate can be shaped into any number of desirableconfigurations to satisfy any number of overall system, geometry orspace restrictions. The matrix can be shaped to different sizes toconform to the necessary structures of different sized patients.

A substrate can also be permeated with a material, for example liquifiedcopolymers (poly-DL-lactide co-glycolide 50:50 80 mg/ml methylenechloride) to alter its mechanical properties. This can be performed bycoating one layer, or multiple layers until the desired mechanicalproperties are achieved.

The substrate can also be treated or seeded with various factors andproteins to control the degradation/absorption of the matrix in thesubject. For instance, if the cells seeded within the substrate areslow-growing, then it is beneficial to maintain the matrix integrity fora long enough period of time to allow the cells enough time toregenerate and grow. On the other hand, if the cells are able to quicklyreproduce and grow, then a short lived substrate could be desirable.Varying the concentration of aprotinin additives, aminocaproic acid,tranxemic acid, or similar fibrinolytic inhibitors or the degree ofchemical cross-linking in the matrix could be used to precisely controlthis variable. The substrate could also be seeded with varying growthfactors such as angiogenesis factor to promote a growth of blood vesselsupon implantation.

VI. Functionalized Matrices

A matrix (e.g., an electrospun matrix, a natural decellularized matrix,or synthetic matrix) can be functionalized by incorporation ofnanoparticles such as quantum dots (QD) coupled to therapeutic orbiological agents. The matrix can also be functionalized to incorporatea contrast enhancing agent (e.g., gadolinium).

In one aspect, the invention pertains to releasing therapeutic orbiological agent in a controlled manner at a target site. This isaccomplished using quantum dots to which the therapeutic/biologicalagent is coupled. Quantum dots are a semiconductor nanocrystal withsize-dependent optical and electronic properties. In particular, theband gap energy of a quantum dot varies with the diameter of thecrystal. The average diameter of the QDs may be between about 1 to about100 nm, between about 10-80 nm, and between about 25-40 nm. The coupledagent can be released by application of energy such as near infrared(NIR) irradiation from a laser source, which causes the bonds betweenthe agent and the QD to break and thus releases the agent. This allowsthe release of the agent to be controlled by triggering its release uponapplication of energy. Quantum dots have been used as photostablebiological fluorescent tags, semiconductors, and thermal therapy. Thehigh transmission, scattering-limited attenuation, and minimal heatingeffects of quantum dots makes these suitable for the coupling oftherapeutic/biological agents. In one embodiment, NIR CdSe quantum dots(Evident Technologies) can be used. These QDs have an optical absorptionrange of 700-1000 nm. NIR energy within this spectral region has beenshown to penetrate tissue at depths up to 23 cm with no observabledamage to the intervening tissue.

A matrix functionalized with a QD coupled to a therapeutic or biologicalagent can be used for controlled release of the therapeutic orbiological agent at a target in the subject. The therapeutic orbiological agent can be released by application of energy at a desiredwavelength such as near infrared irradiation. Due to localized heatingof the QD, ultrastructural changes cause the release of the coupledagent. The release kinetics can be varied according to the type of QDused and the wavelength of irradiation. The release kinetics can also bevaried by altering the intensity and time of irradiation. For example, aQD (e.g., CdSe QD from Evident Technologies) coupled to encapsulatedheparin can be incorporated into an electrospun matrix. Upon applicationof near infrared radiation at a wavelength of 700-1000 nm, the heparinis released in a controlled manner, as described in the examples below.

The studies in the examples section demonstrate the burst release ofheparin over time when quantum dot conjugated heparin nanoparticles wereirradiated by NIR irradiation. This system allows medical personnel totune therapeutic/biological agent release rates post-operatively.

The emission spectra of quantum dots have linewidths as narrow as 25-30nm depending on the size heterogeneity of the sample, and lineshapesthat are symmetric, gaussian or nearly gaussian with an absence of atailing region. The combination of tunability, narrow linewidths, andsymmetric emission spectra without a tailing region provides for highresolution of multiply-sized quantum dots within a system and allowssimultaneous examination of a variety of biological moieties tagged withQDs.

In addition, the range of excitation wavelengths of the quantum dots isbroad and can be higher in energy than the emission wavelengths of allavailable quantum dots. Consequently, this allows the simultaneousexcitation of all quantum dots in a system with a single light source.The ability to control the size of QDs enables one to construct QDs withfluorescent emissions at any wavelength in the UV-visible-IR region.Therefore, the colors (emissions) of QDs are tunable to any desiredspectral wavelength. Furthermore, the emission spectra of monodisperseQDs have linewidths as narrow as 25-30 nm. The linewidths are dependenton the size heterogeneity of QDs in each preparation. In one embodiment,the QDs emit light in the ultraviolet wavelengths. In anotherembodiment, the QDs emit light in the visible wavelengths. In otherembodiments, the QDs emit light in the near-infrared and the infraredwavelengths. Color of the light emitted by the QDs may be size-tunableand excitation energy tunable.

Many QDs are constructed of elements from groups II-VI, III-V and IV ofthe periodic table. They exhibit quantum confinement effects in theirphysical properties, and can be used in the composition of theinvention. Exemplary materials suitable for use as quantum dots include,but are not limited to, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP,GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge, and Siand ternary and quaternary mixtures thereof. The quantum dots mayfurther include an overcoating layer of a semiconductor having a greaterband gap.

Any suitable therapeutic or biological agent such as genetic material,growth factors, cytokines, enzymes can be coupled to the QD. Thetherapeutic or biological agent can be released by the application ofenergy that breaks the bond between the QD and the coupled agent. Theagent may also be released at a specific site as a function ofbiodegradation of the matrix in the surrounding environment over time.

Examples of a therapeutic or biological agent include, but are notlimited to proteins growth factors, antibodies, nucleic acids molecules,carbohydrates, and the like. Growth factors useful in the presentinvention include, but are not limited to, transforming growthfactor-alpha (TGF-α), transforming growth factor-beta (TGF-β),platelet-derived growth factors (PDGF), fibroblast growth factors (FGF),including FGF acidic isoforms 1 and 2, FGF basic form 2 and FGF 4, 8, 9and 10, nerve growth factors (NGF) including NGF 2.5 s, NGF 7.0 s andbeta NGF and neurotrophins, brain derived neurotrophic factor, cartilagederived factor, bone growth factors (BGF), basic fibroblast growthfactor, insulin-like growth factor (IGF), vascular endothelial growthfactor (VEGF), granulocyte colony stimulating factor (G-CSF), insulinlike growth factor (IGF) I and II, hepatocyte growth factor, glialneurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocytegrowth factor (KGF), transforming growth factors (TGF), including TGFsalpha, beta, beta1, beta2, beta3, skeletal growth factor, bone matrixderived growth factors, and bone derived growth factors and mixturesthereof.

Cytokines useful in the present invention include, but are not limitedto, cardiotrophin, stromal cell derived factor, macrophage derivedchemokine (MDC), melanoma growth stimulatory activity (MGSA), macrophageinflammatory proteins 1 alpha (MIP-1 alpha), 2, 3 alpha, 3 beta, 4 and5, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, TNF-alpha, and TNF-beta. Immunoglobulins useful in thepresent invention include, but are not limited to, IgG, IgA, IgM, IgD,IgE, and mixtures thereof. Some preferred growth factors include VEGF(vascular endothelial growth factor), NGFs (nerve growth factors),PDGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.

Other molecules useful as therapeutic or biological agents include, butare not limited to, growth hormones, leptin, leukemia inhibitory factor(LIF), endostatin, thrombospondin, osteogenic protein-1, bonemorphogenetic proteins 2 and 7, osteonectin, somatomedin-like peptide,osteocalcin, interferon alpha, interferon alpha A, interferon beta,interferon gamma, interferon 1 alpha.

Embodiments involving amino acids, peptides, polypeptides, and proteinsmay include any type or combinations of such molecules of any size andcomplexity. Examples include, but are not limited to structuralproteins, enzymes, and peptide hormones. These compounds can serve avariety of functions. In some embodiments, the matrix may containpeptides containing a sequence that suppresses enzyme activity throughcompetition for the active site. In other applications antigenic agentsthat promote an immune response and invoke immunity can be incorporatedinto a construct. In substances such as nucleic acids, any nucleic acidcan be present. Examples include, but are not limited todeoxyribonucleic acid (DNA), and ribonucleic acid (RNA). Embodimentsinvolving DNA include, but are not limited to, cDNA sequences, naturalDNA sequences from any source, and sense or anti-sense oligonucleotides.For example, DNA can be naked (e.g., U.S. Pat. Nos. 5,580,859;5,910,488) or complexed or encapsulated (e.g., U.S. Pat. Nos. 5,908,777;5,787,567). DNA can be present in vectors of any kind, for example in aviral or plasmid vector. In some embodiments, nucleic acids used willserve to promote or to inhibit the expression of genes in cells insideand/or outside the electrospun matrix. The nucleic acids can be in anyform that is effective to enhance its uptake into cells.

The state of the electrospun matrix in relation to the incorporatedtherapeutic or biological agent can be controlled by the couplingchemistry, whether the therapeutic/biological agent is encapsulated, theselection of matrix compounds, the type of QDs used, solvent(s), andsolubility of the matrix compounds in those solvents. These parameterscan be manipulated to control the release of the therapeutic/biologicalagents. It is to be understood that therapeutic/biological agents may beentrapped or entangled within an electrospun matrix, bonded to a matrix,contained within cavities, enclosures, inclusions, or pockets, orstructures of electrospun matrix (e.g. fibers, fibrils, particles) orexternally bound to specific sites on the matrix.

The therapeutic or biological agent can also be entrapped, for exampleencapsulated in a polymer with the QD. The encapsulated QD-agent can bemixed with a solution comprising at least one natural compounds, and atleast one synthetic compound and electrospun into the matrix.

In particular, the therapeutic or biological agent and the nanoparticles(e.g., quantum dot) can be entrapped or encapsulated to produce“nanocapsules.” These nanocapsules containing the agent and thenanoparticle can be produce standard encapsulating techniques.Microencapsulation of agents generally involves three steps: (a)generating microcapsules enclosing the agents (e.g., by forming dropletsof cell-containing liquid alginate followed by exposure to a solution ofcalcium chloride to form a solid gel), (b) coating the resulting gelledspheres with additional outer coatings (e.g., outer coatings comprisingpolylysine and/or polyornithine) to form a semipermeable membrane; and(c) liquefying the original core gel (e.g., by chelation using asolution of sodium citrate). The three steps are typically separated bywashings in normal saline.

Alginates are linear polymers of mannuronic and guluronic acid residues.Monovalent cation alginate salts, e.g., Na-alginate, are generallysoluble. Divalent cations such as Ca²⁺, Ba²⁺ or Sr²⁺ tend to interactwith guluronate, providing crosslinking and forming stable alginategels. Alginate encapsulation techniques typically take advantage of thegelling of alginate in the presence of divalent cation solutions.Alginate encapsulation of agent-nanoparticles generally involvessuspending the agent-nanoparticles to be encapsulated in a solution of amonovalent cation alginate salt, generating droplets of this solution,and contacting the droplets with a solution of divalent cations. Thedivalent cations interact with the alginate at the phase transitionbetween the droplet and the divalent cation solution, resulting in theformation of a stable alginate gel matrix being formed. A variation ofthis technique is reported in U.S. Pat. No. 5,738,876, where the cell issuffused with a solution of multivalent ions (e.g., divalent cations)and then suspended in a solution of gelling polymer (e.g., alginate), toprovide a coating of the polymer.

Another method of microencapsulating agent-nanoparticles is thealginate-polyamino acid technique. Cells are suspended in sodiumalginate in saline, and droplets containing islets are produced.Droplets of cell-containing alginate flow into calcium chloride insaline. The negatively charged alginate droplets bind calcium and form acalcium alginate gel. The microcapsules are washed in saline andincubated with poly-L-lysine (PLL) or poly-L-ornithine (or combinationsthereof); the positively charged poly-l-lysine and/or poly-L-ornithinedisplaces calcium ions and binds (ionic) negatively charged alginate,producing an outer poly-electrolyte membrane. A final coating of sodiumalginate may be added by washing the microcapsules with a solution ofsodium alginate, which ionically bonds to the poly-L-lysine and/orpoly-L-ornithine layer. See U.S. Pat. No. 4,391,909 to Lim et al (allU.S. patents referenced herein are intended to be incorporated herein intheir entirety). This technique produces what has been termed a“single-wall” microcapsule. Preferred microcapsules are essentiallyround, small, and uniform in size. (Wolters et al., J. Appli Biomater.3:281 (1992)).

The alginate-polylysine microcapsules can also then be incubated insodium citrate to solubilize any calcium alginate that has not reactedwith poly-l-lysine, i.e., to solubilize the internal core of sodiumalginate containing the islet cells, thus producing a microcapsule witha liquefied cell-containing core portion. See Lim and Sun, Science210:908 (1980). Such microcapsules are referred to herein as having“chelated”, “hollow” or “liquid” cores. A “double-wall” microcapsule isproduced by following the same procedure as for single-wallmicrocapsules, but prior to any incubation with sodium citrate, themicrocapsules are again incubated with poly-l-lysine and sodiumalginate.

Many alternative techniques used for encapsulating agents are known inthe art and can be used with this invention. U.S. Pat. No. 5,084,350discloses microcapsules enclosed in a larger matrix, where themicrocapsules are liquefied once the microcapsules are within the largermatrix. Tsang et al., U.S. Pat. No. 4,663,286 discloses encapsulationusing an alginate polymer, where the gel layer is cross-linked with apolycationic polymer such as polylysine, and a second layer formed usinga second polycationic polymer (such as polyornithine); the second layercan then be coated by alginate. U.S. Pat. No. 5,762,959 to Soon-Shionget al. discloses a microcapsule having a solid (non-chelated) alginategel core of a defined ratio of calcium/barium alginates, with polymermaterial in the core. U.S. Pat. Nos. 5,801,033 and 5,573,934 to Hubbellet al. describe alginate/polylysine microspheres having a finalpolymeric coating (e.g., polyethylene glycol (PEG)); Sawhney et al.,Biomaterials 13:863 (1991) describe alginate/polylysine microcapsulesincorporating a graft copolymer of poly-l-lysine and polyethylene oxideon the microcapsule surface, to improve biocompatibility; U.S. Pat. No.5,380,536 describes microcapsules with an outermost layer of watersoluble non-ionic polymers such as polyethylene(oxide). U.S. Pat. No.5,227,298 to Weber et al. describes a method for providing a secondalginate gel coating to cells already coated with polylysine alginate;both alginate coatings are stabilized with polylysine. U.S. Pat. No.5,578,314 to Weber et al. provides a method for microencapsulation usingmultiple coatings of purified alginate. U.S. Pat. No. 5,693,514 toDorian et al. reports the use of a non-fibrogenic alginate, where theouter surface of the alginate coating is reacted with alkaline earthmetal cations comprising calcium ions and/or magnesium ions, to form analkaline earth metal alginate coating. The outer surface of the alginatecoating is not reacted with polylysine. U.S. Pat. No. 5,846,530 toSoon-Shiong describes microcapsules containing cells that have beenindividually coated with polymerizable alginate, or polymerizablepolycations such as polylysine, prior to encapsulation.

In one embodiment, heparin is coupled to the nanoparticle and thecontrolled release kinetics of heparin can be monitored. One skilled inthe art will appreciate that the control release kinetics depend on thecapsulation parameters including nanocapsule size, heparin and quantumdot loading, and polymer composition. The mean diameter of thenanocapsules depends on the mixing velocity of the preparation processand viscosity of the preparation media. Nanocapsule size can be reducedby exposing the preparation to sonication over a range of about 30second to about 120 seconds, increasing the sonication intensity fromabout 5 watts to about 20 watts, or by varying the ratios of organicpolymer phase to aqueous heparin phase. Nanocapsule sizes can becharacterized by scanning electron microscopy (SEM), coulter counter,and light scattering.

In one embodiment, the heparin can be conjugated to quantum dots byusing an EDC/NHS chemical method. Various concentrations of heparin(ranging form 10-30 weight % polymer) and quantum dots can be used todetermine optimal loading efficiency.

For polymer encapsulation, FDA approved biodegradable polymers (PLA,PLGA, PCL) can be used for the control of encapsulation and degradationof the nanocapsules in vivo.

The examples show that a burst of heparin release occurs using abroadband infrared (IR) source. Using measured quantities of QD-Heparinnanocapsules (NC) suspended in a physiological buffer, the influence ofvarying wavelengths, intensities, and irradiation times on the releasekinetics can be determined. In one embodiment, the wavelength ofirradiation used on the QD-Heparin can be in the near-infraredwavelength range, such as 700 nm, 800 nm, and 900 nm, using a filteredxenon source. The intensity of irradiation energy can be adjusted inincremental steps from 0 (control), 1 mW/cm², 10 mW/cm², 100 mW/cm², 1W/cm², and 10 W/cm². The irradiation time can also be varied todetermine the optimal irradiation time at each effective powerintensity. The irradiation time can vary from 0 (control), 10, 60, 300,and 600 seconds of exposure.

The encapsulated QD-heparin will be released upon near infra-red (NIR)irradiation due to localized heating of the quantum dots which inducesultrastructural changes in the nanocapsules. The release kinetics willbe varied at the target site by modulating the intensity and time of NIRirradiation to produce a controlled release of heparin. The quantitativemeasurement of heparin released from the nanocapsules can be measuredover time (2, 4, 6, 12, and 24 hours and daily thereafter up to 30 days)and measured for its anti-factor Xa activity with a syntheticchromogenic substrate using a kit Rotachrom (Diagnostica Stago Inc).

In another aspect, the invention pertains to monitoring tissueremodeling a tissue engineered construct. Remodeling that takes placetoo slowly can result in pathologic response of surrounding tissues andcompliance mismatch of the vessel. Rapid remodeling can result inpremature failure of the engineered construct. Magnetic ResonanceImaging (MRI) is a powerful, non-invasive technique that can be usedlong term for monitoring the remodeling process. Nanoparticles (e.g.,QD, image enhancing agents) can be easily bound both to decellularizedmatrices and electrospun matrices, and also embedded within nanofibersof electrospun matrices. The nanoparticles provide high MRI contrast,and due to their small size, will not interfere with normal biologicalprocesses. Organolanthanide complexes containing paramagnetic metalssuch as gadolinium (Gd) have been known to cause distortion in anelectromagnetic field. When the protons in water interact with thisdistorted field, their magnetic properties significantly change suchthat they can be detected by MRI. The Examples demonstrate the enhancedimaging observed using MRI contrast with Gd functionalized nanoparticlesbound to the surface and/or incorporated into the vascular matrices ornanocapsules. Other examples of contrast enhancing agents include, butare not limited to, rare earth metals such as, cerium, samarium,terbium, erbium, lutetium, scandium, barium, bismuth, cerium,dysprosium, europium, hafnium, indium, lanthanum, neodymium, niobium,praseodymium, strontium, tantalum, ytterbium, yttrium, and zirconium.

In one embodiment, the agents are joined to the matrix by peptide bonds.For example, nanoparticles can be incorporated as part of the matrixusing EDC (1-ethyl-3(3-dimethly aminopropyl)carbodiimide) and sulfo-NHS(N-hydrocyl-sulfo-succinimide) to form peptide bonds. Various other knowtechniques can be used as described, for example, in Heumanson,Bioconjugate Techniques, Academic Press San Diego, Calif., 1996, hereinincorporated by reference. For external functionalization, a peptidebond can be created between the matrix and carboxylated gadoliniumnanoparticles using the EDC/sulpho-NHS method to form peptide bondsbetween the carboxylates and amino groups. The quantum dot coupled to atherapeutic/biological agent, a contrast enhancing agent, e.g.,gadolinium, or both, can also be added internally to an electrospunmatrix by incorporating each component into the solution with at leastone natural compound and at least one synthetic compound. For example,solutions containing collagen I, elastin and PLGA, successfullyincorporated the contrast enhancing agent gadolinium uponelectrospinning as described in the Examples. The incorporation of thegadolinium into the matrix can be observed in vitro and in vivo usingdetection methods such as magnetic resonance imaging (MRI). Thus, amatrix functionalized with a contrasting agent allows the degradation ofthe matrix to be monitored.

Any type of functionalization method can be used. Examples of somepossible functionalization chemistries include, but are not limited to,esterification (e.g., with acyl halides, acid anhydrides, carboxylicacids, or esters via interchange reactions), ether formation (forexample, via the Williamson ether synthesis), urethane formation viareactions with isocyanates, sulfonation with, for example,chlorosulfonic acid, and reaction of b-sulfato-ethylsulfonyl aniline toafford an amine derivative that can be converted to a diazo for reactionwith a wide variety of compounds. Such chemistries can be used to attacha wide variety of substances to the electrospun matrix, including butnot limited to crown ethers (Kimura et al., (1983) J. Polym. Sci. 21,2777), enzymes (Chase et al. (1998) Biotechnol. Appl. Biochem., 27,205), and nucleotides (Overberger et al. (1989) J. Polym. Sci. 27,3589).

V. Culturing Cells

The artificial tissue can be created by using allogenic cell populationsderived from the subject=s own tissue. The artificial tissue can also bexenogenic, where cell populations are derived from a mammalian speciesthat are different from the subject. For example, tissue cells can bederived from mammals such as monkeys, dogs, cats, mice, rats, cows,horses, pigs, goats and sheep.

The isolated cells are preferably cells obtained by a swab or biopsy,from the subject's own tissue. A biopsy can be obtained by using abiopsy needle under a local anesthetic, which makes the procedure quickand simple. The small biopsy core of the isolated tissue can then beexpanded and cultured to obtain the tissue cells. Cells from relativesor other donors of the same species can also be used with appropriateimmunosuppression.

Methods for the isolation and culture of cells are discussed byFreshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed.,A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107B126. Cells may beisolated using techniques known to those skilled in the art. Forexample, the tissue can be cut into pieces, disaggregated mechanicallyand/or treated with digestive enzymes and/or chelating agents thatweaken the connections between neighboring cells making it possible todisperse the tissue into a suspension of individual cells withoutappreciable cell breakage. If necessary, enzymatic dissociation can beaccomplished by mincing the tissue and treating the minced tissue withany of a number of digestive enzymes either alone or in combination.These include but are not limited to trypsin, chymotrypsin, collagenase,elastase, and/or hyaluronidase, DNase, pronase, and dispase. Mechanicaldisruption can also be accomplished by a number of methods including,but not limited to, scraping the surface of the tissue, the use ofgrinders, blenders, sieves, homogenizers, pressure cells, or insonatorsto name but a few.

Cell types include, but are not limited to, progenitor cells isolatedfrom the peripheral blood bone that can be induced to differentiate intodifferent cells, stem cells, committed stem cells, and/or differentiatedcells may be used. Also, depending on the type of tissue or organ beingmade, specific types of committed stem cells can be used. For instance,myoblast cells can be used to build various muscle structures. Othertypes of committed stem cells can be used to make organs or organ-liketissue such as heart, kidney, liver, pancreas, spleen, bladder, ureterand urethra. Other cells include, but are not limited to, endothelialcells, muscle cells, smooth muscle cells, fibroblasts, osteoblasts,myoblasts, neuroblasts, fibroblasts, glioblasts; germ cells,hepatocytes, chondrocytes, keratinocytes, cardiac muscle cells,connective tissue cells, epithelial cells, endothelial cells,hormone-secreting cells, cells of the immune system, neurons, cells fromthe heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra,and the like. In some embodiments it is unnecessary to pre-select thetype of stem cell that is to be used, because many types of stem cellscan be induced to differentiate in an organ specific pattern oncedelivered to a given organ. For example, a stem cell delivered to theliver can be induced to become a liver cell simply by placing the stemcell within the biochemical environment of the liver.

Examples also include cells that have been genetically engineered,transformed cells, and immortalized cells. One example of geneticallyengineered cells useful in the present invention is a geneticallyengineered cell that makes and secretes one or more desired molecules.When electrospun matrices comprising genetically engineered cells areimplanted in an organism, the molecules produced can produce a localeffect or a systemic effect, and can include the molecules identifiedabove as possible substances.

Cells may produce substances that inhibit or stimulate inflammation;facilitate healing; resist immunorejection; provide hormone replacement;replace neurotransmitters; inhibit or destroy cancer cells; promote cellgrowth; inhibit or stimulate formation of blood vessels; augment tissue;and to supplement or replace the following tissue, neurons, skin,synovial fluid, tendons, cartilage, ligaments, bone, muscle, organs,dura, blood vessels, bone marrow, and extracellular matrix.

The shape of the extracellular matrix may help send signals to the cellsto grow and reproduce in a specific type of desired way. Other factorsand differentiation inducers may be added to the matrix to promotespecific types of cell growth.

Once the tissue has been reduced to a suspension of individual cells,the suspension can be fractionated into subpopulations from which thecells elements can be obtained. This also may be accomplished usingstandard techniques for cell separation including, but not limited to,cloning and selection of specific cell types, selective destruction ofunwanted cells (negative selection), separation based upon differentialcell agglutinability in the mixed population, freeze-thaw procedures,differential adherence properties of the cells in the mixed population,filtration, conventional and zonal centrifugation, centrifugalelutriation (counterstreaming centrifugation), unit gravity separation,countercurrent distribution, electrophoresis and fluorescence-activatedcell sorting (see e.g. Freshney, (1987) Culture of Animal Cells. AManual of Basic Techniques, 2d Ed., A. R. Liss, Inc., New York, Ch. 11and 12, pp. 137B168). For example, salivary cells may be enriched byfluorescence-activated cell sorting. Magnetic sorting may also be used.

Cell fractionation may also be desirable, for example, when the donorhas diseases such as cancer or tumor. A cell population may be sorted toseparate the cancer or tumor cells from normal noncancerous cells. Thenormal noncancerous cells, isolated from one or more sorting techniques,may then be used for tissue reconstruction.

Isolated cells can be cultured in vitro to increase the number of cellsavailable for seeding into the biocompatible substrate. To prevent animmunological response after implantation of the artificial tissueconstruct, the subject may be treated with immunosuppressive agents suchas, cyclosporin or FK506.

Isolated cells may be transfected with a nucleic acid sequence. Usefulnucleic acid sequences may be, for example, genetic sequences whichreduce or eliminate an immune response in the host. For example, theexpression of cell surface antigens such as class I and class IIhistocompatibility antigens may be suppressed. In addition, transfectioncould also be used for gene delivery. Cells may be transfected withspecific genes prior to seeding onto the biocompatible substitute. Thus,the cultured cells can be engineered to express gene products that wouldproduce a desired protein that helps ameliorate a particular disorder.

The tissue cells grown on the electrospun matrix substrate may begenetically engineered to produce gene products beneficial toimplantation, e.g., anti-inflammatory factors, e.g., anti-GM-CSF,anti-TNF, anti-IL-1, and anti-IL-2. Alternatively, the tissue cells maybe genetically engineered to “knock out” expression of native geneproducts that promote inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or“knock out” expression of MHC in order to lower the risk of rejection.

Methods for genetically engineering cells for example with retroviralvectors, adenoviral vectors, adeno-associated viral vectors,polyethylene glycol, or other methods known to those skilled in the artcan be used. These include using expression vectors which transport andexpress nucleic acid molecules in the cells. (See Geoddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990).

Vector DNA is introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. Suitable methodsfor transforming or transfecting host cells can be found in Sambrook etal. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory press (1989), and other laboratory textbooks.

Once seeded onto the matrix, the cells will proliferate and develop onthe matrix to form a tissue layer. Importantly, because the matrix hasan infra-structure that permits culture medium to reach the tissuelayer, the cell population continues to grow, divide, and remainfunctionally active to develop into a tissue that has a morphology whichresembles the analogous structure in vivo.

It is important to recreate, in culture, the cellular microenvironmentfound in vivo for the particular tissue being engineered. By using amatrix that retains an infra-structure that is similar or the same as anin vivo tissue structure, the optimum environment for cell-cellinteractions, development and differentiation of cell populations, iscreated.

Growth factors and regulatory factors can be added to the media toenhance, alter or modulate proliferation and cell maturation anddifferentiation in the cultures. The growth and activity of cells inculture can be affected by a variety of growth factors such as growthhormone, somatomedins, colony stimulating factors, erythropoietin,epidermal growth factor, hepatic erythropoietic factor (hepatopoietin),and like. Other factors which regulate proliferation and/ordifferentiation include prostaglandins, interleukins, andnaturally-occurring chalones.

The artificial tissue constructs of the invention can be used in avariety of applications. For example, the artificial tissue constructscan be implanted into a subject to replace or augment existing tissue.The subject can be monitored after implantation of the artificial tissueor organ, for amelioration of the disorder.

The artificial tissue can be used in vitro to screen a wide variety ofcompounds, for effectiveness and cytotoxicity of pharmaceutical agents,chemical agents, growth/regulatory factors. The cultures can bemaintained in vitro and exposed to the compound to be tested. Theactivity of a cytotoxic compound can be measured by its ability todamage or kill cells in culture. This may readily be assessed by vitalstaining techniques. The effect of growth/regulatory factors may beassessed by analyzing the cellular content of the matrix, e.g., by totalcell counts, and differential cell counts. This may be accomplishedusing standard cytological and/or histological techniques including theuse of immunocytochemical techniques employing antibodies that definetype-specific cellular antigens. The effect of various drugs on normalcells cultured in the artificial tissue may be assessed.

VI. Use of Matrices

The methods and compositions of the invention can be used for localizeddelivery of therapeutic/biological agents, as well as controlled releaseof such agents at the target site in a subject.

(i) Vascular Constructs

The methods and compositions of the invention can be used to constructblood vessels. One application of the electrospun matrices is in theformation of medium and small diameter vascular constructs. Somepreferred materials for this embodiment are collagen and elastin,especially collagen type I and collagen type III. Examples of vascularconstructs include, but are not limited to coronary vessels for bypassor graft, femoral artery, popliteal artery, brachial artery, tibialartery, radial artery or corresponding veins. The electrospun materialis useful especially when combined with endothelial cells and smoothmuscle cells. More complicated shapes including tapered and/or branchedvessels can also be constructed. A different-shaped mandrel is necessaryto wind the large fibers around or to orient the electrospun polymer.

Some of the complications with vascular matrices are (1) thrombusformation and (2) inability to quantitatively monitor integration of thevascular graft in vivo. Problems with thrombus formation are some of themost difficult challenges resulting in frequent failure of vasculargrafts. Heparin, a powerful anticoagulation agent, is commonlyadministered clinically to avoid thrombus formation. However, systemicuse of heparin carries a certain amount of risk, thus locallyadministered heparin is preferred. The methods and compositions of theinvention can be used to overcome the lack of control of drug release byutilizing quantum dot based nanotechnology. Specifically, theacceleration of release of anticoagulants such as heparin at the targetlocation (vascular graft) by triggering their release from quantum dotsusing NIR energy. This allows the release kinetics of the anticoagulante.g., heparin to be modulated.

The studies shown in the Examples section demonstrated that nearinfrared (NIR) quantum dot conjugated heparin can be successfullyincorporated into the nanoparticles and vascular scaffolds to enable thecontrolled release (or burst release) of heparin over time initiated bynear infrared exposure.

MRI contrasting agents such as gadolinium were also successfullyattached to, or incorporated into the scaffold to enhance visualization.Thus, controlled release of heparin from vascular scaffolds can beachieved using near infrared (NIR) quantum dots and heparin and (2)nanocontrast agents functionalized on, or incorporated into, thevascular scaffold can be used to evaluate and monitor heparin release.

(ii) Tissue Organ Constructs

The methods and compositions of the invention can be used to constructengineered tissue organ constructs, or parts of organ constructs e.g.,heart, heart valves, liver, kidney, and the like. The ability to useelectrospun materials and matrices to bioengineer tissue or organscreates a wide variety of bioengineered tissue replacement applications.Examples of bioengineered components include, but are not limited to,blood vessels, heart, liver, kidney, skeletal muscle, cardiac muscle,and nerve guides. In some embodiments, such matrices are combined withtherapeutic agents that improve the function of the implant. Forexample, antibiotics, anti-inflammatories, local anesthetics orcombinations thereof, can be added to the matrix of a bioengineeredorgan to speed the healing process and reduce discomfort.

(iii) Substance Delivery

The methods and compositions of the invention can be used to deliveryone or more therapeutic agents to a desired location. The presentcompositions can be used to deliver therapeutic agents to an in vivolocation, an in vitro location, or other locations. The presentcompositions can be administered to these locations using any method.Alternatively, an electrospun matrix containing cells can be implantedin a body and used to deliver molecules produced by the cells afterimplantation.

The selection of the therapeutic agent and the method by which the agentis combined with the electrospun material affects the substance releaseprofile. To the extent that the agents are immobilized by theelectrospun matrix, the release rate is more closely related to the rateat which the electrospun material degrades. For example, a therapeuticagent can be electrospun with the matrix and trapped within anelectrospun filaments, in such an instance, the release kinetics aredetermined by the rate at which the electrospun matrix degrades. Inother embodiment, the therapeutic agent can be coupled to a QD andelectrospun into the matrix. In such instances, the release kinetics arecontrolled by the application of irradiation energy that disrupts thecoupling bonds between the therapeutic agent and the QD to release thetherapeutic agent. In other embodiments, the therapeutic agents can beencapsulated within a polymer matrix and the encapsulated therapeuticagent added during the electrospinning process such that theencapsulated therapeutic agents is embedded within the matrix. Underthese circumstances, the release kinetics depend on the rate at whichthe electrospun matrix degrades, as well as the nature and degradationproperties of the encapsulating polymer. In yet other embodiments, thetherapeutic agent can be coupled to a quantum dot and encapsulated andthen electrospun to become embedded within the matrix. Under suchcircumstances, the release kinetics are controlled by the application ofirradiation energy that disrupts the coupling bonds between thetherapeutic agent and the QD to release the therapeutic agent. Theporosity of the electrospun material can also be regulated, whichaffects the rate of release of a substance.

Chemicals that affect cell function, such as oligonucleotides, promotersor inhibitors of cell adhesion, hormones, and growth factors, forexample, can be incorporated into the electrospun matrix and the releaseof those substances from the electrospun matrix can provide a means ofcontrolling expression or other functions of cells in the electrospunmatrix.

Release kinetics in some embodiments are manipulated by cross-linkingelectrospun material through any means. In some embodiments,cross-linking will alter, for example, the rate at which the electrospunmatrix degrades or the rate at which a compound is released from theelectrospun matrix by increasing structural rigidity and delayingsubsequent dissolution of the electrospun matrix. Electrospun matrix canbe formed in the presence of cross-linking agents or can be treated withcross-linking agents after electrospinning. Any technique forcross-linking materials may be used as known to one of ordinary skill inthe art. Examples of cross-linking agents include, but are not limitedto, condensing agents such as aldehydes e.g., glutaraldehyde,carbodiimide EDC (1-ethyl-3(3 dimethyl aminopropyl)), photosensitivematerials that cross-link upon exposure to specific wavelengths oflight, osmium tetroxide, carbodiimide hydrochloride, and NHS(n-hydroxysuccinimide).

The release kinetics of the matrix is also controlled by manipulatingthe physical and chemical composition of the electrospun matrix. Forexample, small fibers of PLGA are more susceptible to hydrolysis thanlarger diameter fibers of PLGA. An agent delivered within an electrospunmaterial composed of smaller PLGA fibers is released more quickly thanwhen prepared within a matrix composed of larger diameter PLGA fibers.

Physical processing of the electrospun matrix is another way tomanipulate release kinetics. In some embodiments, mechanical forces,such as compression, applied to an electrospun matrix hasten thebreakdown of the matrix by altering the crystalline structure of thematrix. The structure of the matrix is thus another parameter that canbe manipulated to affect release kinetics. Polyurethanes and otherelastic materials such as poly(ethylene-co-vinyl acetate), silicones,and polydienes (e.g., polyisoprene), polycaprolactone, polyglycolic acidand related polymers are examples of materials whose release rate can bealtered by mechanical strain.

VII. Storage

A matrix can be stored and used shortly before implantation by seedingit with cells. Many electrospun matrices are dry once they are spun andcan be storage in a dry or frozen state. Storage conditions will dependon the electrospun compounds used and whether a therapeutic agent isincorporated onto or into the matrix. In embodiments where a therapeuticagent is incorporated, the matrix can be stored at temperatures below 0°C., under vacuum, or in a lyophilized state. Other storage conditionscan be used, for example, at room temperature, in darkness, in vacuum orunder reduced pressure, under inert atmospheres, at refrigeratortemperature, in aqueous or other liquid solutions, or in powdered formdepending on the materials in and on the matrix.

The matrices may be sterilized through conventional means known to oneof ordinary skill in the art such as radiation, and heat. The matricescan also be combined with bacteriostatic agents, such as thimerosal, toinhibit bacterial growth. In some embodiments, the compositions can betreated with chemicals, solutions, or processes that confer stability instorage and transport.

Other embodiments and used of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All U.S. patents and other referencesnoted herein for whatever reason are specifically incorporated byreference. The specification and examples should be considered exemplaryonly with the true scope and spirit of the invention indicated by thefollowing claims.

EXAMPLES Example 1 Methods and Materials

(i) Scaffold Preparation

Electrospun nanofiber scaffolds have been developed using a solution ofcollagen type I, elastin, and poly(D,L-lactide-co-glycolide) (PLGA, mol.ratio 50:50, Mw 110,000) (Boeringer-Ingelheim, Germany). Collagen type Ifrom calf skin (Elastin Products Company, Owensville, Mo.), elastin fromligamentum nuchae (bovine neck ligament), (Elastin Products Company,Owensville, Mo.), and PLGA are mixed at a relative concentration byweight of 45% collagen, 40% PLGA, and 15% elastin. The solutes aredissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (99+%) (Sigma ChemicalCompany, St. Louis, Mo.) at a total solution concentration of 15 w/v %(150 mg/mL). High molecular weight PLGA, previously used forelectrospinning tissue scaffolds is added to the solution to increasemechanical strength of the scaffold and increase viscosity and spinningcharacteristics of the solution.

Physically, the electrospinning method requires a high voltage powersupply, a syringe pump, a polymer solution or melt to be spun, and agrounded collection surface. During electrospinning, the groundedmandrel rotates while the stage translates to ensure even deposition offibers onto the mandrel surface. Solutions were electrospun using a highvoltage power supply (Spellman High Voltage, Hauppauge, N.Y.) at 25 kVpotential between the solution tip and the grounded surface. Thesolution was delivered with a 5 mL syringe through an 18 gauge blunt tipneedle at a flow rate of 3.0 mL/hr using a syringe pump. Fibers collectonto a grounded mandrel at a distance of 15 cm from the tip. The mandrelis a 303 stainless steel rod which is rotated at ˜500 rpm. The mandrelsize is initially 4.75 mm to allow for contraction of the graft due tocrosslinking. Uniform scaffolds of 120 mm length were created using 2.4mL of solution. This apparatus is shown schematically in FIG. 1.

Scaffolds were further crosslinked for increased stability and strength,using two crosslinking methods. The scaffolds were soaked for twominutes in 20% dextran solution in phosphate buffered saline prior tocrosslinking to reduce hydration-induced swelling and contraction of thescaffold. The scaffolds were crosslinked by immersion in 1) 1%glutaraldehyde solution and 2) EDC/NHS in MES/EtOH solution for 2 hoursat room temperature. These data show that it is possible to fabricatevascular scaffolds from biological polymers with mechanics and structuresimilar to decellularized scaffolds and native arteries.

FIG. 1. shows the electrospinning apparatus in which fibers deposit ontoa grounded collection surface as solvent evaporates due to increasingsurface area/volume ratio of solution. The electrostatic field causessplaying of solution, and solutions of sufficient viscosity and surfacetension form fibrous mats which adhere to grounded surfaces.

(ii) Cell Seeding

A confluent monolayer of endothelial cells is the most important barrieragainst thrombus formation, and endothelial cell mediated NO productionis important to maintain vascular tone. Cells were seeded with a mouseendothelial cell line MS1 cells. The cells routinely cultured in tissueculture polystyrene flasks at 37° C. under 5% CO₂ atmosphere wereharvested after the treatment with 0.1% trypsin-EDTA. The scaffolds weremounted in tissue culture dishes. After equilibration with PBS, thecells (1×10⁵/mL) were seeded to the scaffolds. The culture medium usedwas DMEM medium containing 10% FBS, and antibiotics. After 2 daysculture, the cell attachment was assessed using scanning electronmicroscopy.

(iii) Microscopy

The relative quantity and distribution of collagen and elastin in avascular scaffold is important to the mechanical properties and functionof the seeded graft. To determine the distribution of components of thescaffolds, histo- and immunohistochemical analyses were performed toidentify collagen and elastin distribution.

(iv) Biocompatibility Testing (Cell Viability and Proliferation)

Long-term viability of cells is necessary for the seeded scaffold toremodel itself into a viable, patent vessel. Standard methods wereemployed to assess viability and proliferation. To test for cellviability, constructs were placed in 24-well plates with approximately100 mg of material per well. Four different types of material weretested for biocompatibility and cell survival, with one negative controlwell with no material: (1) GA-NFS (1% glutaraldehyde crosslinkedelectrospun scaffold); (2) EDC-NFS (EDC-crosslinked electrospunscaffold); (3) nBV (natural blood vessel, decellularized); (4) Latex(latex rubber, positive control).

Endothelial cells were seeded in the wells on a scaffold for testing viathe direct contact method. For cell viability, cell layers were rinsedwith PBS. 0.005% w/v neutral red was added in culture medium. Theneutral red solution was removed after 4 hours incubation at 37° C. with1% acetic acid and 50% ethanol solution by volume was added for dyeextraction, and dye extraction was shaken for 5 minutes. Absorbance wasthen measured at 540 nm using a spectrophotometer. The intensity of redcolor obtained was directly proportional to the viability of the cellsand inversely proportional to the toxicity of the material.

Cell proliferation was tested using the mitochondrial metabolic activityassay. Cell layers were first rinsed with PBS. MTT solution was added at1 mg/mL in PBS containing 1 mg/mL glucose. MTT solution was removedafter 4 hours incubation at 37° C. Dimethyl sulfoxide (DMSO) was used todissolve insoluble formazan crystals, and the absorbance at 540 nm wasmeasured using a spectrophotometer. The intensity of blue color wasdirectly proportional to the metabolic activity of the cell populationsand inversely proportional to the toxicity of the material or extract.

(v) Mechanical Testing

Compliance mismatch is one of the most common causes of vascular graftfailure, resulting in intimal hyperplasia and occlusion. If the scaffoldis too compliant, it may form an aneurysm.

Scaffolds were immersed in a water bath and cannulated at either end.One cannula was connected to a column of water and the other to adrainage tube. The column of water was high enough to create a pressurewithin the vessel-shaped scaffold of 120 mmHg. Water was drained throughthe scaffold in order to lower the pressure in increments of 10 mmHg. Ateach increment, the diameter of the scaffold was recorded using adigital camera. This process was repeated until the pressure was 0 mmHg.

(vi) Axial and Circumferential Segment Testing

Vessels must resist higher stress in the circumferential direction thanin the axial direction. Native vessels adapt their mechanics to thisloading environment. It is important that the electrospun scaffoldsexhibit a mechanical strength at least that of native vessels.Mechanical loading tests were performed on the electrospun vessels inthe axial and circumferential directions using a uniaxial load testmachine (Instron Corporation, Issaquah, Wash.). A short segment from atubular scaffold was clamped at its cut ends for the axial test. Thecrosshead speed was set at 0.5 mm/sec and the test was stopped when thestrain decreased by 10% after the onset of failure. For testing in thecircumferential direction, a ring of material was cut from the scaffold,opened into a strip and then clamped at either end of the strip. Thistest was also performed at a rate of 0.5 mm/sec.

(vii) Burst Pressure Testing

The burst pressure for vascular scaffolds was found by monitoringincreasing pressures within the vessel until failure occurred. Apressure catheter was inserted through a cannulating fixture at one endof the vessel. A 60 cc pressure syringe was inserted through a customcannula at the other end of the vessel. The pressure was increased untilfailure or leakage occurred and the pressure change was recorded.

(viii) Functionalization of Matrices

To functionalize a matrix, EDC (10 mg) and sulfo-NHS (2 mg) were addedto 5 mL (0.05 mg/mL) of carboxylated quantum dots in aqueous solutionunder gentle stirring for 1 hr at room temperature. EDC activatedheparin (30 mg/20 μl) was prepared according to the same EDC and NHSmethod. In order to conjugate quantum dots and heparin, 5 mg PDA wasadded to the activated quantum dots and heparin solutions under stirringfor 2 hr at room temperature. The quantum dot-heparin (QD-heparin)conjugation can be quenched by adding an equal volume of 1 M Tris buffersolution (pH 7.4) and stored in 4° C. (FIG. 2).

(ix) Encapsulation

Microencapsulation of QD-heparin was performed by double emersion.Briefly, 4 mL of internal aqueous phase containing 30 mg QD-heparin and10 mg bovine serum albumin (BSA) as stabilizer was emulsified in 8 mlsolution of 100 mg PLGA and 100 mg PCL in dichloromethane. The solutionwas emulsified by vortexing for 5 minutes at room temperature. This W/Odispersion was diluted to 200 ml of 1% (w/v) aqueous PVA solution understirring for 4 hr at room temperature. The microcapsules were washedseveral times with deionized water and then lyophilized overnight.

(x) Heparin Release Using IR Irradiation of the Quantum Dot

In order to evaluate the burst release of heparin, 0.55 mg of PLGAmicrocapsules containing QD-heparin were suspended in 2 ml of PBS(phosphate buffered saline). The solution was irradiated for 0, 10 and30 min using an AM1.5 solar simulator at 75 mW/cm2. On days 1, 3 and 5,the samples were then cooled to 4° C., centrifuged at 4500 rpm for 20min and filtered (0.45 μm pore size) to remove any microcapsules for theoptical measurements. Luminescence measurements were performed using anargon ion laser (514.5 nm at 400 mW/cm2) as the excitation source andspectra were collected using a CCD spectrophotometer with an integrationtime of 40 sec.

(xi) Mouse Model

Mice (C57BL6) will be obtained from Jackson laboratories, Bar Harbor Me.All experimentation in mice will be performed aseptically under generalanesthesia (ketamine; 45-75 mg/kg and Xylazine; 10-20 mg/kg, IP). Theincision sites are scrubbed with betadine and wiped with alcohol.Analgesia (Buprenorphine 0.05-0.1 mg/kg, SC) is given post-operativelyafter implantation. Prophylactic antibiotic agents (cefazoline 25 mg/kg,sc) are given to the animals at the time of implantation. The preparedblood vessels (2×0.5 cm) will be implanted in the dorsal subcutaneousspace of mice through a minimal longitudinal midline incision with 2implants per animal. The wound will be closed with interruptedabsorbable sutures and the animals will be sacrificed 1, 2, 4, 8, 12, 18and 24 weeks after implantation for analyses. For the collection ofblood samples, mice will be anesthetized and blood will be retrievedinto heparin containing tubes using cardiac puncture and the mice willbe sacrificed thereafter.

(xii) Sheep Model

A total of 120 sheep will be used. The experimental study will consistof 6 different groups of the blood vessels. Each animal will serve asits own control. Animals will be sacrificed at 1, 3, 6, 12, and 18months after implantation. Animals will be monitored at 0, 1, 2, 3, and4 weeks and monthly for grafts implanted greater than one month.

Sheep will be sedated with Ketamine (5 mg/kg, IM), intubated andanesthetized with Isofluorane (1-3%), and placed on a ventilatoradministering Isoflurane for maintenance. Following Duplex ultrasoundimaging of native femoral arteries the groins will be prepped in asterile fashion and antibiotics administered (cefazolin 25 mg/kg, i.v.).A longitudinal incision will be made overlying the superficial femoralartery, which will then be exposed over a length of 6 to 8 cm. Animalswill receive aspirin for 48 hours prior to surgery (80 mg, p.o.) andheparin will be administered immediately prior to implantation (100U/Kg, i.v.). The femoral artery will then be clamped and dividedproximally and an end-to-side anastomosis created between native andengineered artery with running 7-0 Prolene sutures. The distalanastomosis will then be created in a similar fashion and blood flowrestored through the implant. Duplex ultrasound will then be repeatedusing a sterile intraoperative probe cover to establish arterydimensions and blood flow immediately after implantation. Wounds willthen be closed with absorbable sutures and the animals recovered fromanesthesia using Atropine (0.02 mg/kg i.v.) prior return to standardhousing. Post-operative antibiotics will be administered (Cephazoline 25mg/kg/day) for 3 days following the procedure. Analgesia will beadministered (ketoprofen 2 mg/kg) every 6-12 hours for 3 days. Aspirinwill also be administered (80 mg daily) for 7 days orally foranticoagulation. The animals will be sacrificed 1, 3, 6, 12 and 18months after implantation for analyses. At each time point, 6 animalswill be euthanized for analysis.

Example 2 Preparation of Decellularized Organs

The following method describes a process for removing the entirecellular content of an organ or tissue without destroying the complexthree-dimensional infra-structure of the organ or tissue. An organ, e.g.a liver, was surgically removed from a C7 black mouse using standardtechniques for tissue removal. The liver was placed in a flaskcontaining a suitable volume of distilled water to cover the isolatedliver. A magnetic stir plate and magnetic stirrer were used to rotatethe isolated liver in the distilled water at a suitable speed for 24-48hours at 4° C. This process removes the cellular debris and cellmembrane surrounding the isolated liver.

After this first removal step, the distilled water was replaced with a0.05% ammonium hydroxide solution containing 0.5% Triton X-100. Theliver was rotated in this solution for 72 hours at 4° C. using amagnetic stir plate and magnetic stirrer. This alkaline solutionsolubilized the nuclear and cytoplasmic components of the isolatedliver. The detergent Triton X-100, was used to remove the nuclearcomponents of the liver, while the ammonium hydroxide solution was usedto lyse the cell membrane and cytoplasmic proteins of the isolatedliver.

The isolated liver was then washed with distilled water for 24-48 hoursat 4° C. using a magnetic stir plate and magnetic stirrer. After thiswashing step, removal of cellular components from the isolated wasconfirmed by histological analysis of a small piece of the liver. Ifnecessary, the isolated kidney was again treated with the ammoniumhydroxide solution containing Triton X-100 until the entire cellularcontent of the isolated liver was removed. After removal of thesolubilized components, a collagenous three-dimensional framework in theshape of the isolated liver was produced.

This decellularized liver was equilibrated with 1× phosphate buffersolution (PBS) by rotating the decellularized liver overnight at 4° C.using a magnetic stir plate and magnetic stirrer. After equilibration,the decellularized liver was lyophilized overnight under vacuum. Thelyophilized liver was sterilized for 72 hours using ethylene oxide gas.After sterilization, the decellularized liver was either usedimmediately, or stored at 4° C. or at room temperature until required.Stored organs were equilibrated in the tissue culture medium overnightat 4° C. prior to seeding with cultured cells.

Example 3 Electrospun Matrices

An electrospun matrix was formed using the methods outlined inExample 1. A solution of collagen type I, elastin, and PLGA, were used.The collagen type I, elastin, and PLGA were mixed at a relativeconcentration by weight of 45% collagen, 40% PLGA, and 15% elastin.

The resulting fibrous scaffold had a length of 12 cm with a thickness of1 mm. A 2 cm representative sample is depicted in FIG. 3. Thisdemonstrates the feasibility of spinning Type I Collagen and elastininto fibers from nanometer to micrometer diameter using concentrationsfrom 3% to 8% by weight in solution. These results also show that byadding PLGA (Mw 110,000) to the mixture, solutions with higher viscosityand improved spinning characteristics could attained. By increasing thesolution concentration to 15%, thicker, stronger scaffolds were able tobe built while maintaining the collagen and elastin components.

Collagen type I stained positively on the decellularized scaffolds,demonstrating uniform distribution. Elastin distribution within thescaffolds was determined by Movat staining. The electrospun scaffoldswith 15% elastin demonstrated a uniform elastin matrix throughout thescaffold wall. These findings indicate that the matrix content anddistribution of the electrospun scaffolds can be manipulated to achievevarious matrix compositions depending on the need.

Results of biocompatibility assays were calculated as a percentage ofnegative control and both electrospun scaffolds performed similarly tothe decellularized blood vessel. These data suggest that thebiocompatibility of electrospun scaffolds is similar to that ofdecellularized scaffold.

Results of mechanical testing for compliance show a typicalpressure-diameter curve for native vessels, as well as fordecellularized and electrospun scaffolds. The diameter change wasapproximately 5% for native vessels and electrospun scaffolds within thephysiologic pressure range which is consistent with the in vivomechanical behavior of porcine and human arteries (FIG. 4). These datademonstrate that the electrospun scaffolds created have a compliancesimilar to that of a native vessel.

Results of the axial and circumferential mechanical tests fromelectrospun scaffolds tended to exhibit a more isotropic behavior.Strain in the axial and circumferential directions were nearlyequivalent before failure occurred.

The results of burst pressure testing show that the burst pressure forthe electrospun construct was 1,425 mmHg or nearly 12 times systolicpressure. These data suggest that electrospun scaffolds have adequateinitial strength and elasticity to withstand the mechanical environmentwhen being surgically placed in the circulatory environment.

Histological analysis of the explanted vascular scaffolds from miceshowed that there was no evidence of inflammation or tissueencapsulation.

Collectively, these results show that it is possible to control thecomposition of electrospun scaffolds for use as vascular grafts. Higherconcentrations of collagen type I and elastin than previously employed,and mixing with PLGA, result in improved spinning characteristics andstrength of grafts, which resist almost 12× systolic pressure. Scaffoldsalso exhibited compliance characteristics similar to native arteries.Scaffolds had an average fiber diameter of 720 nanometers. EDCcrosslinked scaffolds demonstrate superior cell proliferationcharacteristics to glutaraldehyde crosslinked scaffolds as assessed bymitochondrial metabolic activity assay. Cell viability assays did notdemonstrate as pronounced a difference in crosslinking method. Theseresults are some of the first data on biocompatibility of electrospunscaffolds created with biological polymers and PLGA. This workdemonstrates the promise of electrospinning as a fabrication process forvascular graft scaffolds.

Example 4 Cross-Linking of Electrospun Matrices

This example demonstrates how to increase the strength and stability ofthe electrospun scaffold by chemical cross-linking. The scaffolds weresoaked in 20% dextran solution in phosphate buffered saline prior tocrosslinking to reduce hydration-induced swelling and contraction of thescaffold. The scaffolds were crosslinked by immersion in EDC/NHS inMES/EtOH solution for 2 hours at room temperature. Scanning electronmicrographs of the resulting fibers showed fiber diameters of 500 nm orless and a random orientation of fibers. Atomic force microscopy of thescaffold and a confocal image of nanofibers with an adhering endothelialcell demonstrate the scaffold structure. These data show that it ispossible to fabricate vascular scaffolds from biological polymers withmechanics and structure similar to decellularized scaffolds and nativearteries.

Example 5 Distribution of Collagen and Elastin Content

The relative quantity and distribution of collagen and elastin in avascular scaffold is important to the mechanical properties and functionof the seeded graft. The scaffold composition was assessed usinghistochemical analysis for collagen types I, II, and III, elastin andhematoxylin, and eosin (H&E) staining was also performed.

The levels of collagen type I, II, and III, and elastin fordecellularized matrices and collagen type I and elastin for electrospunmatrices were analyzed using computerized histomorphometric analysis.NIH Image/J Image analysis software (National Institutes of Health,Bethesda, Md.) was used for the analysis.

Immunohistochemical analyses using antibodies specific to collagen typesI, II and III were performed on the decellularized and electrospunscaffolds. The decellularized scaffolds showed similar collagen type Iand III in the vascular media, which corresponds to normal bloodvessels. In this study, 45% collagen type I was used to demonstrate thecontrollability of the scaffold fabrication. Collagen type I stainedpositively on the decellularized scaffolds, however, collagen type IIIstained negatively. Elastin distribution within the scaffolds wasdetermined by Movat staining. Abundant elastin fibers were observed inthe entire decellularized scaffold wall with a prominent distribution inthe serosal and luminal surface. The electrospun scaffolds with 15%elastin demonstrated a uniform elastin matrix throughout the scaffoldwall. These findings indicate that decellularized vascular scaffoldspossess matrices similar to normal vessels and that the matrix contentand distribution of the electrospun scaffolds can be manipulated toachieve various matrix compositions depending on the need.

Histograms of the distribution of color were used to determine relativeamounts of each component from each stain against negative controls. Allvalues were normalized by area for comparison. Amounts of collagen I,elastin, and PLGA were known for electrospun matrices because offabrication parameters. Calibrating the image data for relative amountsof collagen utilized both the normalized areas with negative controls,and was calibrated based on known composition of electrospun matrices.

The results demonstrate the composition of collagen I, II, and III, andelastin, in the decellularized scaffolds as well as componentpercentages in electrospun matrices. These studies show that thecollagen and elastin content of decellularized and electrospun scaffoldsis similar to that of native vessels.

Example 6 Compliance Testing of Scaffolds

Compliance mismatch is one of the most common causes of vascular graftfailure, resulting in intimal hyperplasia and occlusion. If the scaffoldis too compliant, it may form an aneurysm. This example describes how totest for compliance of the scaffolds. Decellularized and electrospunvessel shaped scaffolds were immersed in a water bath and cannulated ateither end. One cannula was connected to a column of water and the otherto a drainage tube. The column of water was high enough to create apressure within the vesselshaped scaffold of 120 mmHg. Water was drainedthrough the scaffold in order to lower the pressure in increments of 10mmHg. At each increment, the diameter of the scaffold was recorded usinga digital camera. This process was repeated until the pressure was 0mmHg. Results show the typical pressure-diameter curve for nativevessels, and the experimental curves for decellularized and electrospunscaffolds. The diameter change was approximately 5% for native andelectrospun and 15% for decellularized scaffolds within the physiologicpressure range which is consistent with the in vivo mechanical behaviorof porcine and human arteries. Thus, both decellularized and electrospunscaffolds have a compliance similar to that of a native vessel.

Example 7 Circumferential and Axial Loading of Decellularized andElectrospun Vessels

Vessels must resist higher stress in the circumferential direction thanin the axial direction. Native vessels adapt their mechanics to thisloading environment. It is important that the decellularized andelectrospun scaffolds exhibit a mechanical behavior similar to nativevessels. Thus, mechanical loading tests were performed on thedecellularized vessels and electrospun vessels in the axial andcircumferential directions using a uniaxial load test machine (InstronCorporation, Issaquah, Wash.). An entire vessel-shaped scaffold wasclamped at its cut ends for the axial test. The crosshead speed was setat 0.5 mm/sec and the test was stopped when the strain decreased by 10%after the onset of failure. For testing in the circumferentialdirection, a ring of material was cut from the scaffold, opened into astrip and then clamped at either end of the strip. This test was alsoperformed at a rate of 0.5 mm/sec. Results of the axial andcircumferential mechanical tests from electrospun scaffolds are shown inFIGS. 5A and 5B, respectively.

The electrospun scaffolds tended to exhibit a more isotropic behavior.Strain in the axial and circumferential directions were nearlyequivalent before failure occurred. In general, the decellularizedconstruct exhibits the orthotropic mechanical behavior that is expectedfrom the known mechanical behavior of arteries. In particular, strain inthe circumferential direction is lower than strain in the axialdirection. This was true for scaffolds prior to and after implantation.

The burst pressure for vascular scaffolds was found by monitoringincreasing pressures within the vessel until failure occurred. Apressure catheter was inserted through a cannulating fixture at one endof the vessel. A 60 cc pressure syringe was inserted through a customcannula at the other end of the vessel. The pressure was increased untilfailure or leakage occurred and the pressure change was recorded. Theresults show that the burst pressure for the decellularized constructwas 1,960 mm Hg or approximately 16 times systolic pressure. The burstpressure for the electrospun construct was 1,425 mm Hg or nearly 12times systolic pressure. We demonstrated that both electrospun anddecellularized scaffolds had adequate strength and elasticity and may besubstitutes for native vessels.

Example 8 Isolation, Characterization and Vessel Seeding of SheepProgenitor EPC and MPC

Progenitor EPC and progenitor muscle cells (MPC) were isolated from 60ml peripheral blood of the internal jugular vein of sheep. TheLleukocyte fraction was obtained by centrifuging on a Histopaque densitygradient. Some of the cells were resuspended in medium and plated onfibronectin coated plates. At 24 hr intervals the floating cells weretransferred to new fibronectin coated plates. EPC were induced by growthin EGM-2 medium that contained VEGF and bFGF. The rest of the cells werecultured in the presence of 10 μM 5-Azacytidin for 24 hours. Thereafterfloating cells were transferred to a new fibronectin coated plate andcultured in myogenic medium (DMEM low glucose containing 20% fetalbovine serum, 10% Horse Serum, 1% Chick Embryo extract and 1%antibiotics) in order to induce MPC. EPC and MPC were cultured for 4-6weeks in order to assume differentiated morphology. Immunohistochemicalanalysis of EPC showed that most of the cells expressed VE cadherin andCD31 but not Desmin. However, MPC showed expression of Vimentin andDesmin but not of VE cadherin. The expression of these markers wasmaintained during culture in vitro. These results indicate that culturedEPC and MPC possess EC and muscle cell phenotype, respectively. EPC werelabeled by PKH 26 green fluorescent dye and MPC were labeled by PKH 27red fluorescent dye. Labeled EPC and MPC were seeded on the luminal andthe outer surfaces of decellularized vessel segments, respectively, inorder to demonstrate the biocompatibility of the decellularized vessel.After 7 days the presence of red and green labeled cells on thedecellularized vessel was noted. In addition, seeded vessels were seededwith a suspension of red-labeled MPC and green labeled-EPC (5×10⁶cells/ml) and cells were allowed to grow for 7 days. The vessels wereembedded in OCT media in order to obtain frozen sections. The sectionswere stained with DAPI. To detect cell nuclei, sections were visualizedusing a fluorescent microscope. Data shows that EPC were maintained onthe luminal side of the scaffold and MPC on the serosal surface.

Example 9 Cell Attachment

A confluent monolayer of endothelial cells is the most important barrieragainst thrombosis formation. Endothelial cell mediated NO production isimportant in maintaining the vascular tone. To examine cell attachment,the decellularized and electrospun vessels were seeded with endothelialcells. Cell attachment was assessed using scanning electron microscopyof scaffolds seeded with a mouse endothelial cell line (MS1). SEMmicrographs reveal a confluent monolayer on the inner surface of boththe decellularized and electrospun vessels at 48 hours. These resultsindicate that endothelial cells form confluent monolayers ondecellularized and electrospun scaffolds.

Example 10 Biocompatibility (Cell Viability and Proliferation)

Long-term viability of cells is necessary for the seeded scaffold toremodel itself into a viable, patent vessel. To test for cell viability,decellularized and electrospun constructs were placed in 24-well plateswith approximately 100 mg of material per well. Four different types ofmaterial were tested for biocompatibility and cell survival, with onenegative control well with no material: (1) GA-NFS (1% glutaraldehydecrosslinked electrospun scaffold); (2) EDC-NFS (EDC-crosslinkedelectrospun scaffold); (3) nBV (natural blood vessel, decellularized);(4) Latex (latex rubber, positive control).

Endothelial cells were seeded in the wells on a scaffold for testing viathe direct contact method. For cell viability, cell layers were rinsedwith PBS. 0.005% w/v neutral red was added in culture medium. Theneutral red solution was removed after 4 hours incubation at 37° C. with1% acetic acid and 50% ethanol solution by volume was added for dyeextraction, and dye extraction was shaken for 5 minutes. Absorbance wasthen measured at 540 nm using a spectrophotometer. The intensity of redcolor obtained was directly proportional to the viability of the cellsand inversely proportional to the toxicity of the material. Results werereported as a percentage of negative control, and both electrospunscaffolds performed similarly to the decellularized blood vessel (FIG.6A).

Cell proliferation was tested using the mitochondrial metabolic activityassay. Cell layers were first rinsed with PBS. MTT solution was added at1 mg/mL in PBS containing 1 mg/mL glucose. MTT solution was removedafter 4 hours incubation at 37° C. Dimethyl sulfoxide (DMSO) was used todissolve insoluble formazan crystals, and the absorbance at 540spectrophotometer. The intensity of blue color was directly proportionalto the metabolic activity of the cell populations and inverselyproportional to the toxicity of the material or extract. Thegluataraldehyde treated matrices show more pronounced differences thanin proliferation assays, with EDC treated scaffolds being similar tonatural blood vessels (FIG. 6B).

Cell viability and proliferation testing was also performed to determinethe effects of various concentrations of gadolinium (Gd) on thescaffolds, on cell survival (FIG. 7). The tests revealed little effectof Gd levels on cell viability or survival. The results indicate thatboth scaffolds can promote cell growth and thus may be used for thebioengineering of vascular grafts.

Example 11 External Functionalization of Matrices

This example describes how to generate matrices with image enhancingagents and quantum dots. In particular, Gd-DPTA and quantum dotfunctionalization of an external scaffold. The scaffold can be anybiocompatible substrate, such as a synthetic PGA matrix, an electrospunmatrix, or a decellularized matrix. At present, no clinically availablevascular graft allows for noninvasive monitoring of the integration ofthe graft in vivo, nor does any graft incorporate anticoagulants intoits structure. A reliable method is needed to attach nanomaterials toscaffolds, e.g., vascular scaffolds, in order to increase functionality,in particular as a material marker and for anticoagulation. CarboxylatedGd and quantum dot (QD) materials were coupled to the surface of boththe decellularized and the electrospun scaffolds using an EDC/sulfo-NHSmethod. Any unreacted material was quenched and removed by rinsing thescaffold with 0.1 M Tris buffer. The liquid from the final washing wascolorless under UV elimination.

Under blacklight illumination the functionalized scaffold showsmulticolor fluorescence. Areas of red-orange emission are from thequantum dots. The pale white color, which is stronger in intensity thanthe control tissue, comes from the Gd containing material that canfluoresce with a pale blue color. The data shows that it is possible toincorporate heparin onto the surface of a scaffold. The scaffolds arealso able to bind Gd.

Example 12 Internal Functionalization of Matrices

This example describes the production of electrospun matrices with imageenhancing agents and therapeutic agents. In particular, Gd-DPTA and QDaddition to the internal electrospun scaffolds. Fabricating vascularscaffolds using electrospinning provides an opportunity to incorporateimage enhancing agents within the bulk material. Solutions were spunsuccessfully containing gadolinium diethylenetriamine pentacetic acid(Gd-DPTA) in HFP at a concentration of 15 mg/mL and with quantum dotsadded at a concentration of 8% by volume from a quantum dot solution of25.5 nmol/mL in toluene. No morphological change was noted in thescaffolds due to the addition of the Gd-DPTA or the QDs. These resultsshow that incorporating nanoparticles into the scaffolds has only aminimal effect on the morphology of the resulting structure.

Example 13 Matrices with Quantum Dots

This example describes how to couple therapeutic agents, such as heparinto the quantum dots (QD). Heparin is a potent anticoagulant agent. Toavoid systemic administration, a method is needed to control the releaseof heparin from the vascular scaffold and to bind the heparin to thescaffold. In this experiment, EDC (10 mg) and sulfo-NHS (2 mg) was addedinto the 5 mL (0.05 mg/mL) of carboxylated quantum dots in aqueoussolution under gentle stirring for 1 hr at room temperature. EDCactivated heparin (30 mg) was prepared according to the same EDC and NHSmethod as described above. In order to conjugate quantum dots andheparin, 5 mg phenylene diamine (PDA) was added to the activated quantumdots and heparin solutions while stirring for 2 hr at room temperature.The quantum dot-heparin (QD-heparin) conjugation can be quenched byadding an equal volume of 1 M Tris buffer solution (pH 7.4) and storedin 4° C.

Microencapsulation of QD-heparin was performed by double emersion.Briefly, 4 mL of internal aqueous phase containing 30 mg QD-heparinconjugation and 10 mg bovine serum albumin emulsified in 8 mL of asolution of 100 mg PLGA (MW; 110,000) and 100 mg PCL (MW; 110,000) inDCM. The solution was emulsified by vortexing for 5 min at roomtemperature. This W/O dispersion was diluted into 200 mL of 1% (w/v)aqueous PVA solution under stirring for 4 hr at room temperature. Themicrocapsules (MCs) were washed several times with deionized water andthen lyophilized overnight. QD-heparin nanocapsules (NC) wereincorporated into scaffolds by placing the functionalized vascularscaffold in 1 wt % PLL in PBS. Vascular scaffolds were immersed in thePLL-nanocapsule solution for 3-4 hours, and lyophilized beforesterilization with gamma irradiation.

A fluorescence image of an isolated microcapsule containing quantum dotsshows that the characteristic fluorescence from the quantum dots used inthis experiment is at 500 nm. The data show that it is possible to bindheparin to quantum dots and encapsulate the bound heparin in abiodegradable polymer, for attachment to the vascular scaffold.

Example 14 Release Kinetics of Heparin: In Vitro Release of Heparin andBurst Release by Irradiation

To assess the effectiveness of quantum dots for controlled delivery ofheparin, the release kinetics of the drug was analyzed following anirradiation burst. In order to evaluate the burst release of heparin,0.55 mg of PLGA microcapsules with QD-heparin were suspended in 2 ml ofbuffered saline solution. The solutions were irradiated for 0.0, 10, and30 min using an AM1.5 solar simulator at 75 mW/cm². On days 1, 3, and 5the samples were then cooled to 4° C. and centrifuged at 4500 rpm for 20min. The solutions were filtered (0.45 m pore size) to remove anymicrocapsules for the optical measurements.

Luminescence measurements were performed using an argon ion laser (514.5nm at 400 mW/cm²) as the excitation source and spectra were collectedusing a CCD spectrophotometer with an integration time of 40 sec.Irradiated samples showed increased luminescence over time indicating a“burst effect”. The kinetic profile of heparin confirms that irradiationinduced the burst release out of functionalized microcapsules. Heparinrelease was monitored by optical analysis (FIG. 8A) and biochemicalanalysis (FIG. 8B). These results indicate that NIR can be used toinitiate the release of heparin from the QD-heparin microcapsules.

Normally, heparin is administered at the site of implantationimmediately following surgery to prevent acute thrombosis. Afterwards,heparin is administrated within the first week twice a day by injection.In order to improve the patient's compliance, heparin could beimmobilized in vascular scaffolds for extended period of time. However,the immobilization of heparin to the scaffolds results in a slow releaseof heparin which is not appropriate for thrombus prevention. Toaccelerate the burst release of heparin, near infrared (NIR) irradiationof the quantum dots bound to heparin can to be used to achieve thisgoal.

Example 15 Determination of the Remaining Heparin in Retrieved VesselImplants from Mice

To assess the effectiveness of heparin in an in vivo model, heparin mustbe evaluated after the explantation of the scaffold. The remainingheparin in functionalized blood vessels (heparin-QD) implanted in micewas determined by toluidine blue staining and most of the heparin isshown to have diffused out of the vessel two weeks after implantation.The heparin content was analyzed by a Rotachrome kit and the dataconfirms that very little heparin remains after two weeks. The data showthat the activity of heparin was successfully prolonged in the scaffoldbeyond its normal 1-2 hour half-life (FIG. 9).

The inflammatory response of quantum dots should be addressed forclinical applications. From the histological analysis of the explantedvascular scaffolds from mice, there was no evidence of inflammation ortissue encapsulation. The data indicate that conjugated heparin had onlya minimal inflammatory response.

Example 16 Evaluation of the Anti-Thrombogenic Properties of HeparinImmobilized Vessels

Although heparin is a powerful anticoagulant, it was important to verifythat this property still exists after immobilization. Two methods ofheparin binding were tested. Thirty milligrams of heparin was incubatedin 20 mM EDC and 10 mM sulfo-NHS in PBS for 2 hours at room temperature,and a 3 mm diameter decellularized scaffold was then immersed inheparin-EDC solution for 2 hours at room temperature. Aftercross-linking, the sample was rinsed in PBS several times to completelyremove residual EDC. Subsequently immobilization of heparin by physicaladsorption was performed using Poly(L-lysine) (PLL): The 3 mm diameterdecellularized scaffold was incubated in 2 mg/mL PLL solution for 2hours at room temperature. The PLL-adsorbed scaffold was immersed in 15mg/mL heparin solution for 1 hour at room temperature. Theanti-thrombogenic property of each method was evaluated using wholeblood from sheep by toluidine blue staining. Immediate coagulation wasobserved from the decellularized scaffold while no significant sign ofcoagulation was found from both EDC and PLL reacted decellularizedscaffolds 36 hours after blood treatment. The heparin-PLL decellularizedscaffold demonstrated the weakest staining which indicated the highestloading of heparin in the scaffold. These results showed thatimmobilized heparin was effective in preventing thrombus.

Example 17 Enhanced MRI Imaging

This example demonstrates the improved imaging observed with gadolinium.In vitro experiments were conducted on cell scaffolds with gadolinium todetermine the improvement in magnetic resonance imaging. Cylindricalcell scaffolds 20 millimeters long with an internal radius of 10millimeters and a outer radius of 14 millimeters were created withdifferent Gd loading concentrations. Cell scaffolds were individuallyplaced in test tubes and submerged in PBS. The four test tubes werearranged left to right in the following order: non-functionalized cellscaffold (control 1), functionalized scaffold (control 2), 1×Gdconcentration cell scaffold, 100×Gd concentration (control 3) and a1000×Gd concentration cell scaffold. (100×designates a concentration insolution during functionalization of 55 mg/kg of Gd-DPTA) Axial T1weighted spin echo images were acquired on a on a GE HealthcareTechnologies magnetic resonance imaging (MRI) 1.5 T TwinSpeed scanner.

The T1 weighted image acquired with a phased array coil and a 200millisecond repetition time (TR) was obtained. Additional imagingparameters are as follows: echo time (TE)=13 ms, slice thickness=0.8 mm,256×128, field of view (FOV)=12 cm×6 cm, number of averages=100, andphase direction was right to left. The cell scaffolding loaded with1000×Gd (right most test tube) is clearly visible compared to controls1, 2, and 3. Samples were washed twice with TRIS buffer and PBS andstored in PBS for 2 weeks prior to imaging.

The previously described experiment was repeated for two different Gdloaded scaffold preparations: surface and volume loading. The scaffoldon the left is a cylindrically shaped scaffold identical to thepreviously described experiment with a surface loaded 1000×Gdpreparation. The scaffold on the right is a planar sheet of scaffoldwith the Gd embedded throughout the electrospun fibers as describedpreviously. The scaffold that had the Gd electrospun into the fibershowed a much higher contrast. The normalized signal intensities of thescaffold for the surface preparation and volume preparation are 1.5±0.2and 2.98±0.35, respectively. The data on MRI Imaging of Gd loadedscaffolds showed that Gd increases MRI contrast in proportion to thelevel of Gd loaded in the scaffold.

Example 18 In Vivo Preliminary Data on Rodents

Although Gd may be maintained in the scaffolds in vitro, it is necessaryto demonstrate that it retains functionality in vivo. This experimentinvestigates the in vivo functionality of the scaffolds. Electrospunvascular scaffolds were implanted subcutaneously in a mouse for twoweeks prior to imaging. Gd was added to one of the vascular scaffolds toenhance its contrast on a T1 weighted image. A sagittal localizer imagewas acquired from the mouse and a T1 weighted coronal image containingthe two scaffolds was prescribed off the sagittal image. The importantimaging parameters of the T1 weighted image are repetition time (TR) 300milliseconds, echo time (TE) 14 milliseconds, and slice thickness 2millimeters. A 50% improvement in image contrast of the Gd scaffoldcompared to the control. These results in a rodent model demonstratethat the characteristics seen in vitro are maintained in vivo.

Example 19 Ex Vivo Preliminary Data on Sheep Engineered Vessels

In vivo results in the rodent model were limited to subcutaneousspecimens. It was necessary to demonstrate similar results in a scaffoldexposed to blood flow in a large animal model. To determine thefeasibility of using the cell seeded scaffolds containing thenanoparticles (heparin conjugated with quantum dots and Gd-DTPA),femoral artery bypass procedures were performed in sheep. Peripheralblood samples were collected, circulating progenitor cells were selectedand differentiated into endothelial and smooth muscle cells in culture.Each cell type was grown, expanded separately and seeded ondecellularized vascular scaffolds containing the nanoparticles (30 mmlong). Nanoparticle containing scaffolds without cells served as acontrol. Under general anesthesia, sheep femoral arteries were imagedwith duplex ultrasonography (B-mode ultrasound and Doppler spectralanalysis) with a high resolution 15 MHz probe (HDI-5000, ATL) prior toscaffold implantation. The femoral artery was exposed through alongitudinal incision over a length of 6 to 8 cm. Aspirin and heparinwere used as anticoagulation and the femoral artery was clamped anddivided proximally. An end-to-side anastomosis was created betweennative and engineered artery. The distal anastomosis was created in asimilar fashion and blood flow restored through the implant followed byligation of native femoral artery between the two anastomoses. Dopplerultrasonography was performed using a sterile probe to establishscaffold dimensions and blood flow after implantation. Wounds wereclosed and the animals recovered from anesthesia prior to 3500 return tostandard housing. Aspirin was administered routinely for 7 days orallyfor anticoagulation.

Duplex ultrasound imaging was performed to determine the presence ofthrombosis, lumen narrowing intimal hyperplasia and graft wallstricture, and graft aneurismal degeneration. Longitudinal andcross-sectional images of the pre- and post operative arterial segmentsshowed a patent lumen 0 with similar peak systolic, end-diastolic andtime averaged velocities as the normal artery. The arterial wallthickness and luminal diameter of the engineered bypass was similar tonative artery. The engineered arterial bypass and the contralateralnormal femoral artery were scanned with MRI. T1 weighted spin echo MRimages were acquired with the following parameters: 256×126 matrix, 12×6mm FOV, 400 ms TR, 13 ms TE, 1 mm slice thickness, and 50 excitations.Average signal intensities of the samples were normalized by thebackground water intensity to account for receiver coil nonuniformities.The normalized intensities were 2.62 and 2.10 for the scaffold andnormal vessel, respectively.

This experiment was repeated at several different TRs and the signalintensity measured for the scaffold and the normal vessels. As expected,the signal intensity for the gadolinium enhanced scaffold is alwaysgreater than the normal vessel. These results confirmed that Gd andheparin loaded decellularized scaffolds maintain patency in a sheepmodel and maintain MRI contrast.

Gadolinium is a MR contrast agent that enhances images primarily bydecreasing the spin-lattice relaxation time (T1) of protons in tissues.Unlike radionuclides, it will remain effective as long as it islocalized in the engineered vessel. These results shown in vitro throughrepeated rinsing of the Gd doped scaffolds and in vivo through imagingof the engineered vessel, that the functionalized Gd nanoparticles arestable in the matrix. Within the first 3 months, approximately 80% ofthe graft will be remodeled. The Gd localized in the matrix willinitially enhance the imaging of the graft. The change in MR signal overtime, as the concentration of Gd decreases with remodeling of thevascular graft, will allow us to quantify the remodeling rates.

Example 20 Histomorphological Characteristics of Bypass Grafts in Sheep

To demonstrate cell attachment on the retrieved engineered vesselsinitially seeded with endothelial and muscle cells, scanning electronmicroscopy was performed 2 weeks after implantation. The implanteddecellularized scaffolds seeded with cells showed a uniform cellattachment on the luminal surface of the engineered artery similar tonormal vessels. The scaffolds without cells failed to exhibit cellattachment. These observations indicate that the cells seeded ondecellularized vascular scaffolds are able to survive and remainattached after surgery.

To assess the histo-morphological characteristics of the retrievedtissue from engineered arterial bypass grafts in sheep, histologicalevaluation was performed. The engineered arterial specimens were fixed,processed and stained with hematoxylin and eosin (H&E) and Movatstaining. The cell seeded engineered grafts contained uniformcellularity throughout the vascular walls. Abundant elastin fibers wereobserved in the entire arterial wall with a prominent distribution inthe serosa and luminal surface. These findings demonstrate that theengineered vessels, seeded with peripheral blood derived progenitorcells differentiated into endothelial and smooth muscle cells, are ableto show an adequate cellular architecture similar to native vessels.

Collectively, these studies show that it is possible to fabricate andfunctionalize both decellularized and electrospun scaffolds with cells(endothelial and smooth muscle) and nanomaterials (quantumdot-conjugated heparin) that are known to have a positive therapeuticbenefit. Moreover, the data shows the successful incorporation ofmolecules (gadolinium) enhancing MRI contrast to monitor the engineeredvessels over time. The combination of functionalization and imagingoffers the potential for making these scaffolds an ideal vascularsubstitute. The matrices are biocompatible, possess the ideal physicaland structural properties, and have been shown to be functional for over4 months in the carotid artery of sheep.

Example 21 To Characterize the Engineered Vascular Grafts

In order for a vessel to function normally, it should have theappropriate structural properties to accommodate intermittent volumechanges. In pathologic conditions, normal vessel function and mechanicalproperties may be compromised. To translate the use of bioengineeredvessels to patients, it is first necessary to confirm that normalvessels are being formed, and that they retain adequate phenotypic andfunctional characteristics over time, especially with growth.

(i) Mechanical Testing

Understanding the mechanical properties of explanted vessels providesinformation about the adaptive remodeling those vessels have undergonewhile in the host animal. Mechanical testing will include arterialelongation (axial and circumferential), compliance, burst pressure,stress relaxation, and creep.

(ii) Phenotypic and Composition Analyses

Histological and immunohistochemical analysis can be performed on theretrieved vascular grafts. Longitudinal and cross sections will be takenfrom the transition zones between native vessels and graft and from therest of the graft. Specimens will be fixed, processed and stained withHematoxylin and eosin (H&E) and Masson's trichrome. Cross-sectionalareas of the adventitia, media, intima and lumen will be measured usingcomputer-assisted analysis of digital images (NIH Image Software). Inaddition to cross-sectional analysis of the engineered artery body, aseparate analysis will be performed for the anastomoses region betweennative and engineered arteries. The proximal and distal anastomoses willbe fixed in formalin, embedded in paraffin, and then cut incross-section for analysis of lumen caliber and artery wall thickeningin step-sections spanning each anastomosis. In parallel, quantitation ofthrombus formation will be performed using H&E staining. The phenotypiccharacteristics of the retrieved tissues will be determined over time.

To determine the degree of endothelial and smooth muscle content of thebioengineered vessels over time, in comparison to normal tissues,multiple molecular markers will be probed immunocytochemically and withWestern blot analyses, as described above. These markers will includeAnti-Desmin and Anti-Alpha Smooth Muscle Actin, which specificallydetects smooth muscle cells. Endothelialization will be evaluated byanti-von-Willebrand factor anti-CD-31 and anti-VEGF receptor, KDR,antibodies, which stain EC specifically. Cell proliferation andapoptosis in engineered arteries will be determined by BrdUincorporation and TUNEL staining.

The composition and distribution of extracellular matrix components,such as collagen and elastin, are important for the normal function ofblood vessels. While the collagen network is responsible for tensilestrength, elastin is important for the elastic recovery of the vessel.Therefore, an assessment of the collagen and elastin content anddistribution of the retrieved tissues over time will be performed withhistological and quantitative biochemical assays. To determine whetherthe retrieved vessels possess normal concentrations of collagen andelastin, as compared to normal controls, the total collagen and elastincontent per unit wet weight of the retrieved tissue samples will bemeasured quantitatively using the Sircol collagen and the Fastin elastinassay systems (Accurate Chemical & Scientific Corporation, Westbury,N.Y.). To determine the anatomical distribution of collagen within theengineered vessels, as compared to controls, Immunocytochemicallocalization of collagen types I, II and III will be performed usingspecific monoclonal antibodies (Southern Biotechnology Associates, Inc.,Birmingham, Ala.) and with the elastin-specific stain, Movat.

(iii). Physiological Analysis

The ability to synthesize vasoactive agents such as Nitric Oxide (NO)will further determine the functionality of the engineered vascularscaffolds. There is increasing evidence on the importance of NO invascular hemostasis. NO contributes to resting vascular tone, impairsplatelet activation, and prevents leukocyte adhesion to the endothelium.

Briefly, guinea pig thoracic aorta will be harvested, the endotheliumlayer removed by gentle rubbing and cut into 5-mm segments. Each segmentwill be suspended between 2 tungsten stirrups for measurement ofisometric tension. The vessel segments placed in an organ chamber with10 ml Kreb's buffer solution at 37° C. with a mixture of 5% CO₂, 15% O₂and a balance of N2. Each vessel (2-3 cm in length) is tied to a 21 Gneedle, which was attached to plastic IV tubing and placed above theorgan chamber with the fresh aortic segment. The segments will becontracted with 80 mM KCl Kreb's buffer in a stepwise fashion to obtaina resting tension of 4 g. After resting for 90 minutes, the segments arecontracted in response to prostaglandin F2α up to a final concentrationof 10-7M and until a stable contraction of approximately 50% of maximumKCl-induced contraction is achieved. Vasoactive agents and antagonistsare then added using an infusion pump through the vessels to induce NOproduction. Doses of the vasoactive agents between 10⁻⁷-10⁻³ M will betested and dose-response curves will be constructed.

1. An electrospun matrix having a three-dimensional ultrastructure of interconnected fibers and pores to permit cell attachment and further comprising nanoparticles incorporated within the matrix.
 2. The matrix of claim 1, wherein the nanoparticles further comprises quantum dots made of a material selected from the group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge, Si and combinations thereof.
 3. The matrix of claim 2, wherein quantum dot comprises a CdSe quantum dot.
 4. The matrix of claim 1, wherein the nanoparticle is coupled to a therapeutic agent.
 5. The matrix of claim 4, wherein the therapeutic agent is selected from the group consisting of growth factors, proteins, antibodies, nucleic acids molecules, and carbohydrates.
 6. The matrix of claim 4, wherein the therapeutic agent is a growth factor selected from the group consisting of transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain derived neurotrophic factor, cartilage derived factor, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), and skeletal growth factor.
 7. The matrix of claim 4, wherein the therapeutic agent is heparin.
 8. The matrix of claim 4, wherein the therapeutic agent and the nanoparticle are encapsulated in a polymer.
 9. The matrix of claim 8, wherein the therapeutic agent is released from the nanoparticle by application of radiation.
 10. The matrix of claim 9, wherein the radiation causes localized heating of the nanoparticles which induces structural changes in the polymer to release the therapeutic agent.
 11. The matrix of claim 9, wherein the wavelength of the radiation is between 700-1000 nanometers.
 12. The matrix of claim 1, wherein the matrix further comprises collagen.
 13. The matrix of claim 11, wherein the collagen is selected from the group consisting of collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, collagen VII, collagen VIII, collagen IX, and collagen X.
 14. The matrix of claim 1, wherein the matrix further comprises elastin.
 15. The matrix of claim 1, wherein the matrix further comprises a synthetic polymer.
 16. The matrix of claim 15, wherein the synthetic polymer is selected from the group consisting of poly(lactic acid) polymers, poly(glycolic acid)polymers, poly(lactide-co-glycolides) (PLGA), poly(urethanes), poly(siloxanes) or silicones, poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethyl methacrylate), poly(N-vinyl pyrrolidone), poly(methyl methacrylate), poly(vinyl alcohol) (PVA), poly(acrylic acid), poly(vinyl acetate), polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene glycol), poly(methacrylic acid), polylactic acid (PLA), polyglycolic acids (PGA), nylons, polyamides, polyanhydrides, poly(ethylene-co-vinyl alcohol) (EVOH), polycaprolactone, poly(vinyl acetate), polyvinylhydroxide, poly(ethylene oxide) (PEO), and polyorthoesters or a co-polymer formed from at least two members of the group.
 17. The matrix of claim 15, wherein the synthetic polymer comprises poly(lactide-co-glycolides) (PLGA).
 18. The matrix of claim 1 further comprising at least one natural component and at least one synthetic polymer component.
 19. A method of controlling the release of a therapeutic agent at a target site, comprising: providing a matrix having a three-dimensional ultrastructure of interconnected fibers and pores to permit cell attachment and further comprising nanoparticles incorporated within or on a the matrix, wherein the nanoparticle is coupled to a therapeutic agent; delivering the matrix to a target site; and releasing the therapeutic agent at the target site to thereby provide a controlled release of the therapeutic agent.
 20. The method of claim 19, wherein the matrix is selected from the group consisting of an electrospun matrix, a decellularized matrix, and a synthetic polymer matrix.
 21. The method of claim 19, wherein the nanoparticles further comprises quantum dots made of a material selected from the group consisting of ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, GaN, GaP, GaAs, GaSb, InP, InAs, InSb, AlS, AlP, AlAs, AlSb, PbS, PbSe, Ge, Si and combinations thereof.
 22. The method of claim 21, wherein quantum dot comprises a CdSe quantum dot.
 23. The method of claim 19, wherein the therapeutic agent is selected from the group consisting of growth factors, proteins, antibodies, nucleic acids molecules, and carbohydrates.
 24. The method of claim 23, wherein the therapeutic agent is a growth factor selected from the group consisting of transforming growth factor-alpha (TGF-α), transforming growth factor-beta (TGF-β), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain derived neurotrophic factor, cartilage derived factor, bone growth factor (BGF), basic fibroblast growth factor, insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte colony stimulating factor (G-CSF), hepatocyte growth factor, glial neurotrophic growth factor (GDNF), stem cell factor (SCF), keratinocyte growth factor (KGF), and skeletal growth factor.
 25. The method of claim 23, wherein the therapeutic agent is heparin.
 26. The method of claim 19, wherein the therapeutic agent and the nanoparticle are encapsulated in a polymer.
 27. The method of claim 19, wherein the step of releasing the therapeutic agent comprises application of radiation.
 28. The method of claim 27, wherein the radiation causes localized heating of the nanparticle which induces structural changes in the polymer to release the therapeutic agent.
 29. The method of claim 27, wherein the wavelength of the radiation is between 700-1000 nanometers.
 30. The method of claim 19, wherein the matrix is an electrospun matrix and the nanoparticles are incorporated within the matrix during electrospining of the electrospun matrix.
 31. The method of claim 19, wherein the matrix is a decellularized matrix or a synthetic polymer matrix and the nanoparticles are incorporated on the matrix by reacting the surface of the matrix with the nanoparticles. 